Oligopeptidic compounds and uses thereof

ABSTRACT

Disclosed is A method of treatment of a disorder or condition where it is desirable to inhibit the growth of cells, or a method of treatment which involves cytostatic therapy by administering an oligopeptidic compound to a subject in need thereof. The oligopeptidic compound is capable of interacting with proliferating cell nuclear antigen (PCNA) The compound comprises a PCNA interacting motif which is: which is: [K/R]-[F/Y/W]-[L/I/V/A/M]-[L/I/V/A/M]-[K/R] (SEQ ID NO. 28). 
     The oligopeptidic compound has 9-70 subunits and at least one signal sequence. The signal sequence is a nuclear localization signal sequence and/or a cell penetrating signal sequence. In the compound a PCNA interacting motif is N-terminal to a signal sequence.

FIELD OF THE INVENTION

The present invention relates to novel agents, pharmaceuticalcompositions, and their use in therapy, particularly any therapy whereit is desirable or advantageous to reduce or prevent the proliferationor growth of cells such as in the treatment of hyperproliferativediseases or indeed any condition which requires or is responsive tocytostatic therapy. The invention is based on the identification ofnovel interactions between the proliferating cell nuclear antigen (PCNA)and various proteins involved in DNA repair, maintenance and cell cycleregulation and the consequent identification of a novel pentapeptidemotif responsible for such interactions, which we have termed APIM.Accordingly, the present invention more particularly relates to peptidesor mimetics thereof comprising such a motif and which are capable ofinteracting with PCNA, pharmaceutical compositions comprising suchagents, and the use of such agents in therapy, particularly therapiesinvolving the reduction or prevention of cell proliferation, asindicated above. Also provided are therapeutic methods which comprisethe use of an agent comprising a PCNA interacting motif, preferably incombination with a cytostatic agent.

BACKGROUND OF THE INVENTION

Human and animal cells are exposed to a variety of causes of DNA damagesuch as reactive oxygen species, UV light, x-rays and endogenous orexogenous cytostatic agents.

Cytostatic agents are agents which inhibit or suppress cellular growthand/or multiplication (proliferation/replication), for example bydamaging DNA or by interfering with the cellular replication machinery.Alkylating agents are a class of cytostatic agents, some of which areused clinically or for research purposes.

Alkylating agents cause DNA damage by modifying bases at N or O atoms.The type of damage depends on the type of agent, with most agentscausing a specific DNA modification. DNA damage includes alkylationadducts and inter strand cross-links which may lead to miscoding duringreplication and/or replication blocks followed by double strand breaksor translesion synthesis.

Human and animal cells possess various DNA repair systems including baseexcision repair, nucleotide excision repair and mismatch repair. Anexample is the human DNA oxidative demethylase, hABH2, which reconverts3-methylcytosine (3meC) into cytosine and 1-methyladenine (1meA) intoadenine by oxidative demethylation.

Close co-ordination between DNA repair and cell-cycle regulated DNAreplication is essential for genome integrity. It is important that inthe presence of damage, DNA replication is halted until the damage hasbeen repaired, otherwise mutations arise and are propagated. One proteinknown to be involved in both DNA replication and DNA repair isproliferating cell nuclear antigen (PCNA).

PCNA is member of the sliding clamp family of proteins which arefunctionally conserved from bacteria to higher eukaryotes, and whosemain function is to provide replicative polymerases with the highprocessivity needed for duplication of the genome. In live S-phasecells, PCNA tagged with green fluorescent protein (GFP) forms distinctfoci representing sites of replication. It can therefore be used as anS-phase marker.

Numerous proteins involved in cellular processes such as DNA repair,chromatin assembly, epigenetic and chromatin remodelling,sister-chromatid cohesion, cell cycle control and survival are localisedin so-called replication factories which contain more than a dozenreplication forks. Many of these proteins interact with PCNA through theconserved PCNA interacting peptide sequence called the PIP-box(QxxL/I/MxxF/DF/Y), wherein x can be any amino acid. An alternative PCNAbinding motif called the KAx box was identified using a peptide displaylibrary, but this motif has not been verified to be important for PCNAinteractions in vivo.

Various proteins interact with PCNA and some of these proteins,including hABH2, have been shown to co-localise with PCNA in replicationfoci. However, co-localisation in itself does not imply that there isany direct or indirect interaction between co-localising proteins.Indeed, the absence in hABH2 of PCNA-binding motifs such as the PIP-boxor the KAx box would suggest that hABH2 does not interact with PCNA.

SUMMARY OF THE INVENTION

In work leading up to the present invention, the inventors havesurprisingly found that various proteins interact with PCNA via a novelPCNA interacting motif. In one of these proteins, hABH2, this motif islocated at the N-terminus. The inventors established that this motif isboth essential and sufficient for interaction with PCNA (see Example 1).

As explained in more detail in the Examples below, to explore thefunction of this PCNA interacting motif in DNA repair of alkylatingdamage, cell lines expressing a recombinant peptide comprising the motifwere exposed to various doses of MMS (methyl methanesulfonate) which isan S_(N)2 alkylating agent that causes the formation of 3-methylcytosineand 1-methyladenine. It was found that expression of the recombinantpeptide comprising the motif sensitised cells to DNA damage caused byMMS, indicating that the recombinant peptide comprising the motifcompetitively inhibited the interaction between PCNA and hABH2.

Other agents including BCNU, temozolomide (TZM) and mitomycin c (MMC)which cause other kinds of DNA damage were also tested and to theirgreat surprise, the inventors found that the recombinant peptidecomprising the motif also sensitised cells to the damage caused by theseagents. This was entirely unexpected, because BCNU is anO⁶-chloroethylating agent that mainly leads to interstrand crosslinks aswell as some mono-base cyclic adducts (1,N(6)ethenoadenine), TZM isreported to be an O⁶G methylating agent, and MMC causes interstrandcross links via N-alkylation of guanine in CpGs, and hABH2 does notrepair these types of DNA damage. Instead, there are other enzymes whichrepair this kind of damage, for example the damage by TZM is repaireddirectly by O⁶-methylguanine-DNA transferase (MGMT). These findingsindicate that the recombinant peptide comprising the motif does notmerely inhibit the interaction between hABH2 and PCNA, and that otherproteins may be involved, namely that other proteins may interact withPCNA via the novel motif.

The inventors also found that expression of the recombinant peptidecomprising the motif increased the cytotoxic effect of cytostatic agents(specifically MMS) beyond that observed in cell lines from ABH2knock-out mice, i.e. mice which do not possess ABH2, further indicatingthat the recombinant peptide comprising the motif has a wider-rangingeffect, and likely inhibits other proteins in addition to hABH2.

These surprising findings have led the inventors to propose atherapeutic use for a peptide comprising a PCNA binding motif.

The novel PCNA binding motif of the invention, termed APIM, has beencharacterised and may be defined as follows:

(SEQ ID NO: 1) X₁X₂X₃X_(3′)X_(1′),

wherein X₁ and X_(1′) are independently selected from the group of basicamino acids, X₂ is a lipophilic amino acid and X₃ and X_(3′) areindependently selected from the group of uncharged, preferably non-polaramino acids.

A peptide (or oligopeptidic compound) capable of interacting with PCNAmay contain or comprise such a peptide (or sequence) motif. Thus, anoligopeptidic compound capable of interacting with PCNA and comprisingsuch a motif is disclosed herein and may represent certain aspects ofthe present invention.

For example, in one embodiment, the present invention may provide anoligopeptidic compound which is capable of interacting with PCNA andwhich comprises the motif X₁X₂X₃X_(3′)X_(1′) (SEQ ID NO: 1), wherein X₁and X_(1′) are independently selected from the group of basic aminoacids, X₂ is a lipophilic amino acid and X₃ and X_(3′) are independentlyselected from the group of uncharged, preferably non-polar amino acids,wherein the oligopeptidic compound is further characterised by at leastone of the following:

(i) the oligopeptidic compound comprises at least 11 amino acids orequivalent subunits;

(ii) X₂ is not phenylalanine;

(iii) the oligopeptidic compound comprises at least one D-amino acid;

(iv) the oligopeptidic compound comprises at least one signal sequence,namely a sequence which directs the oligopeptidic compound to aparticular location, for example into a cell (e.g. a cell penetratingsequence which directs the oligopeptidic compound into a cell) and/orinto a particular cellular compartment (e.g. a nuclear localisationsignal which directs the oligopeptidic compound into the nucleus); and

(v) the oligopeptidic compound comprises the motif[K/R]-F-[L/I/V]-[L/I/V]-[K/R](SEQ ID NO: 27).

In particular, in such an embodiment the oligopeptidic compoundcomprises a nuclear localisation signal sequence. In another embodimentthe oligopeptidic compound comprises a cell penetrating sequence (cellpenetrating peptide). In a still further embodiment the oligopeptidiccompound comprises a cell penetrating sequence and a nuclearlocalisation sequence.

Thus, it will be seen that in such embodiments the compound of theinvention may take the form of a construct containing (i.e. comprising)an oligopeptidic compound which comprises a PCNA interacting motif asdefined above, together with at least one signal sequence. In thisaspect the invention may accordingly be seen to provide a constructcomprising an oligopeptidic compound which is capable of interactingwith PCNA and which comprises the motif X₁X₂X₃X_(3′)X_(1′) (SEQ ID NO:1), wherein X₁ and X_(1′) are independently selected from the group ofbasic amino acids, X₂ is a lipophilic amino acid and X₃ and X_(3′) areindependently selected from the group of uncharged, preferably non-polaramino acids, together with at least one signal sequence.

As noted above the novel motif of the invention has been determined tomediate the interaction of an oligopeptidic compound (e.g. peptide) orprotein containing such a motif with PCNA.

The interaction may be direct or indirect, and may involve directbinding of the motif to the PCNA, or the motif may bind indirectly, forexample binding may be mediated by another molecule. This reference to“PCNA-interacting” or “PCNA-binding” can thus include any form ofinteraction, and both direct and indirect binding.

Any reference herein to a “motif” should be understood to meanX₁X₂X₃X_(3′)X_(1′) as defined herein.

Preferably, X₁ and X_(1′) are independently selected from lysine (K),arginine (R), histidine (H), ornithine (Orn), methyllysine (MeK) andacetyllysine (AcK), and more preferably K, R and H, or K and R;

X₂ is preferably an aromatic amino acid, more preferably it is selectedfrom phenylalanine (F), tryptophan (W), tyrosine (Y),tert.-butylglycine, cyclohexylalanine, tert.-butylphenylalanine,biphenylalanine and tri tert-butyltryptophan (in certain embodimentsthis list may exclude F), particularly F, W and Y, or W and Y, F and Y,or F and W or in specific embodiments X₂ may be F, or W or Y;

X₃ and X_(3′) are preferably aliphatic amino acids and may for examplebe independently selected from leucine (L), isoleucine (I), valine (V),alanine (A) methionine (M) and norleucine (Nor);

Preferably, X₃ and X_(3′) are not both A, more preferably X₃ and X_(3′)are selected from L, I, V and M, even more preferably from L, I and V.

The binding of the motif to PCNA may in certain embodiments be improvedwhen X₂ is W or Y. Thus, in one embodiment, X₂ is not F. However, asindicated above, in other embodiments it may be F.

Thus, the invention may provide an oligopeptidic compound comprising themotif [K/R]-[F/Y/W]-[L/I/V/A/M]-[L/I/V/A/M]-[K/R](SEQ ID NO: 28),wherein said oligopeptidic compound is capable of interacting with PCNA.

In another embodiment the motif may be defined as:[K/R]-[Y/W]-[L/I/V/A/M]-[L/I/V/A/M]-[K/R](SEQ ID NO: 29).

In another embodiment the motif may be defined as:[K/R]-[F/Y/W]-[L/I/V/A]-[L/I/V/A]-[K/R] (SEQ ID NO: 30).

In another embodiment the motif may be defined as:[K/R]-[Y/W]-[L/I/V/A]-[L/I/V/A]-[K/R] (SEQ ID NO: 31).

In another embodiment the motif may be defined as:[K/R]-[F/W]-[L/I/V/A/M]-[L/I/V/A/MHK/R] (SEQ ID NO: 32).

In another embodiment the motif may be defined as:[K/R]-[F/W]-[L/UV/A]-[L/UV/A]-[K/R] (SEQ ID NO: 33).

In another embodiment the motif may be defined as:[K/R]-[F/Y/W]-[L/I/V]-[L/I/V]-[K/R] (SEQ ID NO: 34).

In another embodiment the motif may be defined as:[K/R]-[F/Y/W]-[L/I/V]-[L/I/V]-[K/R](SEQ ID NO: 35).

In yet another embodiment the motif may be defined as:[K/R]-[Y/W]-[L/I/V]-[L/I/V]-[K/R](SEQ ID NO: 36).

In yet another embodiment the motif may be defined as:[K/R]-F-[L/I/V]-[L/I/V]-[K/R](SEQ ID NO: 37).

The oligopeptidic compound is preferably an isolated compound.

In a preferred embodiment, the oligopeptidic compound has or comprisesthe sequence RFLVK (SEQ ID NO: 2). In other preferred embodiments, theoligopeptidic compound has or comprises a sequence selected from KFLLR(SEQ ID NO: 3), KYLLR (SEQ ID NO: 4), KWLLR(SEQ ID NO: 5), KYILR (SEQ IDNO: 6), KYVLR (SEQ ID NO: 7), RFLLR (SEQ ID NO: 8), RYLLR (SEQ ID NO:9), RWLLR (SEQ ID NO: 10), RYILR (SEQ ID NO: 11), RYVLR (SEQ ID NO: 12),RFLIR (SEQ ID NO: 13), RYLVR (SEQ ID NO: 14) RWLMR (SEQ ID NO: 15),RYVLR (SEQ ID NO: 16), RYVIR (SEQ ID NO: 17), RWLVK (SEQ ID NO: 18),RYLVK (SEQ ID NO: 19), RWLIK (SEQ ID NO: 20), RWIVK (SEQ ID NO: 21),RWVVK (SEQ ID NO: 22), RWAVK (SEQ ID NO: 23), RYVVK (SEQ ID NO: 24),RYLIK (SEQ ID NO: 25) or RYLMK (SEQ ID NO: 26). These specific sequencesare listed by way of example and they are not intended to be limiting onthe scope of the present invention.

In one preferred embodiment, the oligopeptidic compound of the inventionalso comprises a signal sequence which targets the motif to a specificcell type, facilitates entry of the compound into a cell, and/orlocalises the compound to a specific intracellular compartment,preferably the nucleus.

The signal sequence may thus be viewed as any sequence which acts tolocalise, or alternatively put, to direct, translocate or transport, theoligopeptidic compound to any desired location e.g. to any desiredcellular or subcellular location. In preferred embodiments the desiredlocation is a cell (i.e. the inside of a cell) and/or the nucleus of acell.

Thus the signal sequence may be a sequence which acts to transport theoligopeptidic compound into a cell, or across a cell membrane (i.e. intothe interior of a cell). It may thus be a so-called “cell penetrating”sequence (or more particularly “cell penetrating peptide”) also known inthe art as a protein transduction domain (PTD) or protein transductionsequence.

Accordingly, as noted above a preferred embodiment of the invention is aconstruct comprising (i) an oligopeptidic compound comprising an APIMmotif (i.e. a PCNA-interacting motif) as defined herein, and (ii) a cellpenetrating sequence (more particularly a cell penetrating peptide).

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be further described with reference to thefollowing non-limiting Examples and Figures in which

FIG. 1 contains confocal microscope images which show that hABH2 andPCNA co-localise and that the 10 N-terminal amino acids of hABH2 arenecessary and sufficient or this co-localisation. Various hABH2constructs (full length hAHB2 having residues 1-261, truncated hABH2having residues 11-261 and an N-terminal fragment of HABH2 consisting ofresidues 1-10) labelled with EYFP were tested for co-localisation withECFP-labelled PCNA (see Example 1). The left lane shows cellstransfected with hABH2 alone, while the three remaining lanes show cellsco-transfected with a hABH2 construct and PCNA.

FIG. 2 is a graph showing the results of FRET analysis. Normalised FRET(N_(FRET)) measurements are shown between EYFP (yellow fluorescentprotein)/ECFP (cyan fluorescent protein) (Lane 1, background due todimerisation of the tags), EYFP-PCNA/ECFP-PCNA (Lane 2, positive controlbecause PCNA binds to PCNA), hAHB2-EYFP/ECFP-PCNA (Lane 3) and 1-10NhABH2-EYFP/ECFP-PCNA

(Lane 4).

FIG. 3A-FIG. 3D contain graphs showing the effect of various cytostaticagents on cells expressing hABH2₁₋₁₀-EYFP, or expressing EYFP as acontrol. Treatments are shown on the left, untreated cells on the right.FIG. 3A shows treatment carried out with 10 μM MMS. FIG. 3B showstreatment carried out with 40 μM BCNU. FIG. 3C shows treatment carriedout with 1 μM MMC. FIG. 3D shows treatment carried out with 600 μM TMZ.Treatments were carried out for 4 days (FIGS. 3 A-D respectively). Datapresented is from one representative out of at least 3 experiments, thecell growth at different doses was tested in 8 parallel wells.

FIG. 4 shows a sequence alignment of the 10 N-terminal amino acids ofABH2-homologs from Homo sapiens (NP 001001655.1; SEQ ID NO: 135), BosTaurus (NP 001019687.1; SEQ ID NO: 136), Rattus norvegicus (XP 222273.3;SEQ ID NO: 137), Mus musculus (NP 778181.2; SEQ ID NO: 138), Gallusgallus (XP 415188.2; SEQ ID NO: 139) and Strongylocentrotus purpuratus(XP 797704.1; SEQ ID NO: 140) using Clustal W. The sequences wereobtained from a public database.

FIG. 5 shows an alignment of the sequences of the proteins identified inExample 4 from various different species. TFIIS elongation factor likeprotein TFIIS (Homo sapiens/1-10, SEQ ID NO: 141; Mus musculus/1-10, SEQID NO: 142; Gallus gallus/1-10, SEQ ID NO: 143; and Xenopustropicalis/1-10, SEQ ID NO: 144); General transcription factor II,TFII-I, isoform y (Homo sapiens/428-437, SEQ ID NO: 145; Musmusculus/428-437, SEQ ID NO: 146; Xenopus laevis/369-378, SEQ ID NO:147; Homo sapiens/533-542, SEQ ID NO: 148; Mus musculus/533-542, SEQ IDNO: 149; Xenopus laevis/473-482, SEQ ID NO: 150; Homo sapiens/638-647,SEQ ID NO: 151; Mus musculus/638-647, SEQ ID NO: 152; Xenopuslaevis/577-586, SEQ ID NO: 153; Homo sapiens/800-809, SEQ ID NO: 154;Mus musculus/800-809, SEQ ID NO: 155; and Xenopus laevis/732-741, SEQ IDNO: 156); DNA topoisomerase II alpha, TopoT II a (Homo sapiens/965-974,SEQ ID NO: 157; Mus musculus/964-973, SEQ ID NO: 158; Gallusgallus/966-975, SEQ ID NO: 159; and Xenopus laevis/963-972, SEQ ID NO:160).

FIG. 6A-FIG. 6H present graphs showing the results of cytotoxicityassays with various peptides as described in Example 7. FIG. 6A showspeptides MDR26-0 (SEQ ID NO. 100). FIG. 6B shows MDR26-3 (SEQ ID NO.103). FIG. 6C shows MDR26-4 (SEQ ID NO. 104). FIG. 6D shows MDR26-8 (SEQID NO. 106). FIG. 6E shows MDR26-7 (SEQ ID NO. 105). FIG. 6F showsMDR26-72-0 (SEQ ID NO. 112). FIG. 6G shows MDR26-72-01 (SEQ ID NO. 115).FIG. 6H shows MDR34 (SEQ ID NO. 116). HeLa cells were seeded into 96well plates (6000 cells/well) and incubated for 3 hours. Various dosesof peptides were added to the wells in presence (filled diamonds) orabsence of serum (filled square) in the media. After 1 h equal volumemedia with 10% or 20% serum (to serum free media) were added to thewells. The cells were incubated for 48 hours before measurement of cellsurvival by the MTT assay. Graphs show cell growth (OD750 nm) againstconcentration of peptide (04).

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

Cell penetrating peptide (CPP) technology has developed greatly overrecent years and a wide variety of cell penetrating peptides are knownand described in the art and indeed a range of such peptides arecommercially available. Cell penetrating peptides may vary greatly insize, sequence and charge, and indeed in their mechanism of function(which is presently not known for some peptides and not fully elucidatedfor others), but share the common ability to translocate across theplasma membrane and deliver an attached or associated moiety (theso-called “cargo”) into the cytoplasm, or even in some cases thenucleus, of a cell. CPPs are thus peptide-based delivery vectors.

CPPs may be derived from naturally-occurring proteins which are able totranslocate across cell membranes such as the Drosophila homeoboxprotein Antennapedia (a transcriptional factor), viral proteins such asthe HIV-1 transcriptional factor TAT and the capsid protein VP22 fromHSV-1, and or they may be synthetically-derived, e.g. from chimericproteins or synthetic polypeptides such as polyarginine. As noted above,there is not a single mechanism responsible for the transduction effectand hence the design of CPPs may be based on different structures andsequences. Cell penetrating peptides are reviewed in Jarver et al. 2006Biochimica et Biophysica Acta 1758, pages 260-263 and Table 2 belowlists various representative peptides. U.S. Pat. No. 6,645,501 furtherdescribes various cell penetrating peptides which might be used.

TABLE2 CPP SEQUENCE REFERENCE Antp Class PenetratinRQIKIWFQNRRMKWKK (SEQ ID NO: 38) Bolton (2000) Eur. J. Neuro. 12:287Penetratin RRMKWKK (SEQ ID NO: 39) US 6472507 derivativesNRRMKWKK (SEQ ID NO: 40) EP4855781 QNRRMKWKK (SEQ ID NO: 41) WO 97/12912FQNRRMKWKK (SEQ ID NO: 42) RREKWKK (SEQ ID NO: 43)RRQKWKK (SEQ ID NO: 44) KRMKWKK (SEQ ID NO: 45) RKMKWKK (SEQ ID NO: 46)RROKWKK (SEQ ID NO: 47) RRMKQKK (SEQ ID NO: 48) RRMKWFK (SEQ ID NO: 49)RORKWKK (SEQ ID NO: 50) RRMWKKK (SEQ ID NO: 51) RRMKKWK_ (SEQ ID NO: 52)(using standard single amino acid notation,ornithine (O), diaminobutyric acid (B), norleucine (N)) D-Penetratinrqikiwfqnrrmkwkk (SEQ ID NO: 53) Rouselle, C. et al. (2000) Mol. Pharm57:679 Protegrin Class Pegelin RGGRLSYSRRRFSTSTGR (SEQ ID NO: 54)Rouselle, C. (SynB) et al. (2000) Mol. Pharm 57:679 HIV-TAT ClassHIV-TAT GRKKRRQRRRPPQ (SEQ ID NO: 55) Vives E.J Biol, Chem 1997,272:16010 Snyder (2004) PLOS 2:186 47-57OF YGRKKRRQRRR (SEQ ID NO: 56)Potocky et al. HIV-TAT (2003) JBC VP22 DAATATRGRSAASRPTERPRAPARSASRPRRVDElliott g. Cell  (SEQ ID NO: 57) 1997, 88:223- 233 Amphipathic peptidesMAP KLALKLALKALKAALKLA (SEQ ID NO: 58) Morris MC., Nat Biotechnol. 2001,19:1173-1176 Transportan GWTLNSAGYLLGKINLKALAALAKKIL Pooga M, (SEQ ID NO: 59) FASEB J 1998, 12:67- 77 Transportan-AGYLLGKINLKALAALAKKIL (SEQ ID NO: 60) Soomets U, 10 Biochim Biophys Acta2000, 1467:165-176 KALA WEAKLAKALAKALAKHLAKALAKALKACEA Oehike J., (SEQ ID NO: 61) Biochim Biophys Acta 1998, 1414:127-139 Pep-1KETWWETWWTEWSQPKKKRKV Wyman  (SEQ ID NO: 62) Biochemistry 1997,36:3008-3017 Pep-2 KETWFETWFTEWSQPKKKRKV  (SEQ ID NO: 63) MPGGALFLGFLGAAGSTMGAWSQPKSKRKV Wagstaff KM  (SEQ ID NO: 64) Curr MedChem 2006, 13:1371-1387 Vectocell VKRGLKLRHVRPRVTRMDV (SEQ ID NO: 65)Coupade peptides SRRARRSPRHLGSG* (SEQ ID NO: 66) (2005)LRRERQSRLRRERQSR* (SEQ ID NO: 67) Biochem. J. GAYDLRRRERQSRLRRRERQSR 407(SEQ ID NO: 68) *indicate saddition of cys for conjugation to cargo Wr-TKETWWETWWTEWWTEWSQ-GPG-rrrrrrrr Kondo (2004) transporter (SEQ ID NO: 69)Mol. Can. r = D-enantiomer arginine Thera 1623 Other peptides R7RRRRRRR (SEQ ID NO: 70) Rothbard et al., Nat. Med 6 (2000) 1253-1257

Antennapedia-derived CPPs (Antp class) represent a class of particularinterest, based around the 16 amino acid Penetratin sequence as shown inTable 2, which corresponds to the third loop of antennapedia protein andwas shown to be responsible for translocation of the protein. Penetratinhas been extensively developed as a delivery vehicle, includingparticularly for pharmaceutical use, and a wide range of Penetratinderivatives and modified sequences have been proposed and described.Reference may be made in particular to WO 91/1891, WO 00/1417, WO00/29427, WO 2004/069279 and U.S. Pat. No. 6,080,724. Thus, the 16 aminoacid sequence of Penetratin may be modified and/or truncated, or thepeptide may be chemically-modified or retro-, inverso- or retro-inversoanalogues may be made whilst retaining cell-penetrating activity.

Another group of cell penetrating peptides which may advantageously beused are based on the HIV-TAT sequence and HIV-TAT and fragments thereofrepresent a preferred class of CPPs for use according to the presentinvention. Various TAT-based CPPs are described in U.S. Pat. No.5,656,122. An exemplary HIV-TAT peptide as used in the Examples below isRKKRRQRRR (SEQ ID. No. 71) but it will readily be appreciated thatlonger or shorter TAT fragments may be used.

As mentioned above no particular structural features or sequence motifsare common to all CPPs. However, various classes of CPPs may beidentified by particular features, such as for example peptides whichare amphipathic and net positively charged. Other groups of CPPs mayhave a structure exhibiting high α-helical content. Another group may bepeptides characterised by a high content of basic amino acids. CPPs maythus be or may comprise oligomers of basic amino acids such as argininee.g. 5 to 20, 6 to 15 or 6 to 12 R residues e.g. R₇ (SEQ ID NO: 70), R₈(SEQ ID NO: 72) or R₁₁ (SEQ ID NO: 73) or QSR₈ (SEQ ID NO: 74).

Proline-rich amphipathic peptides are another class of CPP and suchpeptides characterised by the presence of pyrrolidine rings fromprolines are described in Pujals et al. 2008 Advanced Drug DeliveryReviews 60, pages 473-484.

Other successfully developed CPPs include pVEC (Elmquist et al. 2003Biol. Chem 384, pages 387-393; Holm et al. 2005 Febs Lett. 579, pages5217-5222) and calcitonin-derived peptides (Krauss et al. 2004 Bioorg.Med. Chem. Lett., 14, pages 51-54).

Commercially available CPPs include Chariot, based on the Pep-1 peptide(Active Motif, France), the Syn-B vectors based on the protegrin peptidePG-1 (System, France), and Express-si Delivery based on the MPG peptidefrom Genospectra, USA.

In addition to publically available and reported CPPs, novel orderivative CPP peptides may be designed and synthesized based on knownor reported criteria (e.g. known CPP sequences or features such as basicamino acid content, α-helical content etc as discussed above).Additionally, randomly-designed or other peptides may be screened forCPP activity, for example by coupling or attaching such a peptidecontaining a reporter molecule e.g. a detectable label or tag such as afluorescent tag to the desired cargo (an oligopeptidic compoundaccording to the present invention) and testing to see if the constructis translocated across the cell membrane, for example by adding thesepeptides to live cells followed by examination of cellular import e.g.using confocal microscopy.

Indeed, whilst it is generally the case that a CPP will penetrate orenter virtually any cell type, it may in some cases be observed thatsuccessful or efficient delivery may be dependent, or may varydepending, on the precise nature of the cargo (e.g. cargo peptidesequence) and/or the CPP used. It would be well within the routine skillof the person skilled in the art to determine optimum peptide sequencesand combinations etc, and to test and/or modify cargo and/or CPPsequence or structure etc.

As mentioned above, the signal sequence which may be comprised withinthe oligopeptidic compounds (or constructs) of the invention may be asignal peptide which directs the compound (or construct) into aparticular sub-cellular compartment, and in particular into the nucleus.Nuclear localisation signals (NLSs) are again well known in the art andwidely described in the literature and any known or functional NLS maybe used.

Accordingly, a further preferred embodiment of the invention is aconstruct comprising (i) an oligopeptidic compound comprising an APIMmotif (i.e. a PCNA-interacting motif) as defined herein, and (ii) anuclear localisation signal.

An NLS may vary in length and/or sequence and a wide range of specificNLS sequences have been described. In general, however, it has beenfound that peptides comprising positively charged amino acids (notablylysine (K), arginine (R) and/or histidine (H)) may function as an NLS.An exemplary NLS may thus be a peptide of e.g. 4-20, more particularly4-15, 4-12, 4-10 or 4-8 amino acids, wherein at least 4 amino acids (andmore particularly at least 60, 70, 75, 80, 85, or 90% of the amino acidresidues in the NLS peptide) are positively charged amino acids,preferably selected from K, R or H. Such an exemplary NLS may forexample have or comprise the sequence RKRH (SEQ ID NO: 75).

Nuclear localisation signals, including both actualexperimentally-determined and predicted or proposed NLS sequences, andstrategies for identifying NLSs are described in Lange et al., J. Biol.Chem. 2007, 282(8), 5101-5105; Makkerh et al., Current Biology 1996,6(8), 1025-1027; Leslie et al., Methods 2006, 39, 291-308; and Lusk etal. Nature Reviews MCB 2007, 8, 414-420.

A classical NLS consists of either one (monopartite) or two (bipartite)stretches of basic amino acids. A monopartite NLS may be exemplified bythe SV40 large T antigen NLS (¹²⁶PKKKRKV¹³² [SEQ ID NO: 76]) and abipartite NLS by the nucleoplasmin NLS (¹⁵⁵ KRPAATKKAGQAKKKK¹⁷⁰ [SEQ IDNO: 77]). The monopartite NLS consensus sequence K-[K/R]-X-[K/R](SEQ IDNO: 78) has been proposed and accordingly an NLS according to thepresent invention may in one embodiment comprise or consist of such aconsensus sequence (where X is any amino acid).

A representative bipartite NLS according to the invention may have thesequence KR-[X]₅₋₂₀-KKKK (SEQ ID NO: 79), e.g. KR-X₁₀-KKKK (SEQ ID NO:80) (where X is any amino acid).

An alternative exemplary bipartite NLS may take the form RKRH-[X]₂₋₁₀-KK(SEQ ID NO: 81) e.g. RKRH-X₂-KK(SEQ ID NO: 82), for example RKRH-II-KK(SEQ ID NO: 83).

The oncoprotein c-myc NLS differs from classical NLSs in that only 3 of9 amino acid residues are basic(PAAKRVKLD [SEQ ID NO: 84]), indicatingthat an NLS need not necessarily conform to the consensus or classicalsequences given above. Makkerh et al (supra) describe NLS sequences inwhich a cluster of basic amino acids (e.g. KKKK [SEQ ID NO: 85]) isflanked by neutral and acidic residues, for example PAAKKKKLD (SEQ IDNO: 86).

Other possible NLS sequences which may be given by way of exampleinclude: PKKKRKVL (SEQ ID NO: 87), KKKRK (SEQ ID NO: 88), KKKRVK (SEQ IDNO: 89), KKKRKVL (SEQ ID NO: 90) and RKKRKVL (SEQ ID NO: 91). Any NLSwhich is a derivative of a known NLS e.g. the SV40, nucleoplasmin, UNG2or c-myc NLS may be used.

A putative, proposed or predicted NLS sequence can be tested for NLSactivity using principles and assays known and described in the art. Forexample a candidate NLS sequence may be attached to the desired cargo(in this case an oligopeptide according to the invention as definedherein) and the construct may be provided with a detectable reportermolecule (e.g a tag or label which may be visualised, for example afluorescent label) and contacted with a test cell. Distribution of theconstruct in the cell may then be determined.

Thus, by way of summary, the skilled person will be aware of suitablesignal sequences, but by way of example the following are mentionedherein. Examples of cell-penetrating peptide sequences includePenetratin™, a 16-amino acid peptide corresponding to the third helix ofthe homeodomain of Antennapedia protein, R rich tags such asR6-Penetratin (in which arginine-residues were added to the N-terminusof Penetratin) and derivatives of the HIV Tat protein such asGRKKRRQRRRPPQQ (SEQ ID NO: 92). Examples of nuclear localisationsequences include the SV40 protein derivative KKKRK (SEQ ID NO: 93).

A preferred construct according to the present invention comprises (i)an oligopeptidic compound comprising an APIM motif as defined herein,(ii) a nuclear localisation signal, and (iii) a cell penetrating signalsequence.

The separate elements or components of a construct according to thepresent invention may be contained or presented in any order, butpreferably in the orders indicated above (e.g APIM oligopeptidiccompound-CPP; APIM oligopeptidic compound-NLS; APIM oligopeptidiccompound-NLS-CPP).

Furthermore, an oligopeptidic compound or construct of the invention maycontain more than one PCNA-interacting motif. Thus, alternatively put, aconstruct according to the present invention may contain more than oneoligopeptidic compound comprising a PCNA-interacting motif. A constructor oligopeptidic compound may for example contain 1-10, e.g. 1-6, or 1-4or 1-3 or one or two motifs. Within a construct also containing a signalsequence, such motifs may be spaced or located according to choice e.gthey may be grouped together, or they may be separated by signalsequence elements e.g. motif-NLS-motif-CPP; ormotif-NLS-motif-motif-CPP; or motif-motif-NLS-CPP etc.

The components or elements of a construct according to the invention maybe attached or linked to one another in any desired or convenient wayaccording to techniques well known in the art. Thus, the components orseparate parts may be linked or conjugated chemically e.g. using knownchemical coupling technologies or the constructs may be formed as asingle whole using genetic engineering techniques e.g. techniques forforming fusion proteins, or they may simply be synthesized as a wholee.g. using peptide synthesis techniques.

The separate parts or components may be linked directly to each other orthey may be linked indirectly by means of one or more linker (or spacer)sequences. Thus, a linker sequence may interspace or separate two ormore individual parts of a construct or separate motif elements in anoligopeptidic construct. The precise nature of the linker sequence isnot critical and it may be of variable length and/or sequence, forexample it may have 0-40, more particularly 0-20, 0-15, 0-12, 0-10, 0-8,or 0-6, 0-4 or 0-3 residues e.g 1, 2 or 3 or more residues. By way ofrepresentative example the linker sequence, if present, may have 1-15,1-12, 1-10, 1-8, 1-6 or 1-4 residues etc. The nature of the residues isnot critical and they may for example be any amino acid, e.g a neutralamino acid, or an aliphatic amino acid, or alternatively they may behydrophobic, or polar or charged or structure-forming e.g. proline. Arange of different linker sequences have been shown to be of use,including short (e.g. 1-6) sequences of neutral and/or aliphatic aminoacids.

Exemplary linker sequences thus include any single amino acid residuee.g. A, I, L, V, G, R, Q, T, or W, or a di-, tri- tetra- penta- orhexa-peptide composed of such residues.

As representative linkers may be mentioned I, II, IL, R, W, WW, WWW,RIL, RIW, GAQ, GAW, VAT, IILVI (SEQ ID NO: 94), IILVIII (SEQ ID NO: 95)etc.

The linkers between different elements may be the same or different.

In one embodiment, there is provided an oligopeptidic compound having orcomprising the sequence MDRWLVKRILVATK (SEQ ID NO: 96) orMDRWLVKRILKKKRKVATKG (SEQ ID NO: 97).

Other representative compounds (or more particularly constructs) of theinvention include MDRWLVKGAQPKKKRKVLRQIKIWFQNRRMKWKK (SEQ ID NO: 98),MDRWLVKGAWKKKRVKIIRKKRRQRRRK (SEQ ID NO: 99),MDRWLVKGAWKKKRKIIRKKRRQRRRG (SEQ ID NO: 100),MDRWLVKGAWKKKRKIIRKKRRQRRRK (SEQ ID NO: 101),MDRWLVKRIWKKKRKIIRKKRRQRRRK (SEQ ID NO: 102),MDRWLVKWWWKKKRKIIRKKRRQRRRK (SEQ ID NO: 103),MDRWLVKWWRKRHIIKKRKKRRQRRRK (SEQ ID NO: 104),MDRWLVKRIWKKKRKIIRRRRRRRRRRRK (SEQ ID NO: 105),MDRWLVKRIWKKKRKIIRQIKIWFQNRRMKWKK (SEQ ID NO: 106),MDRFLVKGAWRKRHIIKKRKKRRQRRRK (SEQ ID NO: 107),MDRWLVKWKKKRKIRRRRRRRRRRRK (SEQ ID NO: 108), MDRWLVKWKKKRKIRKKRRQRRRK(SEQ ID NO: 109), MDRWLVKWRKRHIRKKRRQRRRK (SEQ ID NO: 110),Ac-MDRWLVKGAWRKRHIRKKRRQRRRK (SEQ ID NO: 111),Ac-MDRWLVKWKKKRKIRRRRRRRRRRR (SEQ ID NO: 112),Ac-MDRALVKWKKKRKIRRRRRRRRRRR (SEQ ID NO: 113),Ac-MDRWLVKKKKRKRRRRRRRRRRRK (SEQ ID NO: 114), Ac-MDRWLVKKKKRKRRRRRRRRRRR(SEQ ID NO: 115), MDRWLVKRIWKKKRKIIRWLVKWWWRKKRRQRRRK (SEQ ID NO: 116),KRRRQRRKKRIIKRKKKWWWKVLWRDM (SEQ ID NO: 117).

Oligopeptidic compounds having sequences as set out in SEQ ID NOS. 98 to117 are shown in Table 3 in Example 6 below, which shows the separatecomponents making up the constructs (i.e. motif-containing sequence,linker, NLS, CPP, etc.) Thus, it will be seen that SEQ ID NOS. 98 to 117represent constructs comprising at least one motif-containing sequence,an NLS and a CPP, in some cases linked by linker sequences which mayvary in sequence, as specified. SEQ ID NO. 117 (RI-MDR26-3) is aretro-inverse peptide made up of D-amino acids. NLS sequences based onthe SV40 or UNG2 NLS sequences are used, and CPP sequences based onPenetratin, HIV-TAT or an R-rich peptide.

In a further aspect, the invention provides a nucleic acid moleculeencoding a peptide having or comprising (e.g. of) SEQ ID NO: 1 asdefined above. Also provided is the complement of such a nucleic acidmolecule. Preferably, the nucleic acid molecule comprises a promotersequence operably linked to the sequence encoding a peptide having orcomprising (e.g. of SEQ ID NO: 1. In a preferred embodiment, the nucleicacid molecule also encodes a signal sequence as defined above.

The nucleic acid molecule of the invention comprises at least 15nucleotides and preferably no more than 800 nucleotides, more preferablyno more than 700, 650, 600, 550, 500, 450, 400, 350, 300, 250, 200, 150,100 or 50 nucleotides. The nucleic acid molecule is preferably anisolated molecule.

A further aspect relates to a vector comprising a nucleic acid moleculeas defined herein. The vector may also contain further elementstypically found in a vector such as an origin of replication, aselectable marker such as antibiotic resistance, and/or a multiplecloning site. The vector may further be an expression vector, and maycomprise further elements, e.g. transcriptional and/or translationalcontrol or regulatory elements for expression of the nucleic acidmolecules. Such control elements, e.g. promoters, ribosome bindingsites, enhancers, terminators etc. are well known and widely describedin the art.

The vector may for example be a plasmid or a virus, preferably it isselected from a retrovirus, an adenovirus and an adenovirus-associatedvirus.

In another aspect, there is provided a recombinant host cell containinga nucleic acid molecule and/or vector as described above. The host cellis an animal cell, preferably a mammalian cell, most preferably a rat,murine or human cell.

By “recombinant” is meant that the nucleic acid molecule and/or vectorhas been introduced into the host cell. The host cell may or may notnaturally contain an endogenous copy of the nucleic acid molecule, butit is recombinant in that an exogenous or further endogenous copy of thenucleic acid molecule and/or vector has been introduced.

In a further aspect, there is provided a pharmaceutical compositioncomprising an oligopeptidic compound as defined herein, a nucleic acidmolecule as defined herein and/or a vector as defined herein, togetherwith a pharmacologically (or pharmaceutically) acceptable excipient.

The excipient may include any excipients known in the art, for exampleany carrier or diluent or any other ingredient or agent such as buffer,antioxidant, chelator, binder, coating, disintegrant, filler, flavour,colour, glidant, lubricant, preservative, sorbent and/or sweetener etc.

The excipient may be selected from, for example, lactic acid, dextrose,sodium metabisulfate, benzyl alcohol, polyethylene glycol, propyleneglycol, microcrystalline cellulose, lactose, starch, chitosan,pregelatinized starch, calcium carbonate, calcium sulfate, dextrates,dextrin, dextrose, dibasic calcium phosphate dihydrate, tribasic calciumphosphate, magnesium carbonate, magnesium oxide, maltodextrin, mannitol,powdered cellulose, sodium chloride, sorbitol and/or talc.

The pharmaceutical composition may be provided in any form known in theart, for example as a tablet, capsule, coated tablet, liquid,suspension, tab, sachet, implant, inhalant, powder, pellet, emulsion,lyophylisate, effervescent, spray, salve, emulsion, balm, plaster or anymixtures thereof. It may be provided e.g. as a gastric fluid-resistantpreparation and/or in sustained action form. It may be a form suitablefor oral, parenteral, topical, rectal, genital, subcutaneous,transurethral, transdermal, intranasal, intraperitoneal, intramuscularand/or intravenous administration and/or for administration byinhalation.

In a representative embodiment, the pharmaceutical composition is in aform suitable for liposomal administration, so preferably liposomescontaining the pharmaceutical composition are provided. When liposomesare used, it may not be necessary to include a further excipient, soalso provided are liposomes containing an oligopeptidic compound asdefined herein, a nucleic acid molecule as defined herein and/or avector as defined herein.

Using a database search, the inventors discovered the presence of thenew PCNA binding motif in more than 200 other proteins, many of whichare involved in DNA repair, maintenance and cell cycle regulation, e.g.transcription, replication, phosphorylation, ubiquitinylation,translesion synthesis, sister chromatid cohesion and cell cycleregulation (see Table 1 and Example 4). These proteins include thefollowing

a protein of unknown function which contains the conserved N-terminaldomain I found in TFIIS elongation factor, an important protein for theprogression of stalled transcription, so the inventors named itTFIIS-like protein. This protein contains the motif within its 7N-terminal amino acids.

the multifunctional transcription factor TFII-I, which is critical forcell cycle control and proliferation. Cells over-expressing TFII-I haveincreased persistence of γ-H2AX foci (a marker for DNA double strandbreaks), suggesting a role for TFII-I in DNA repair. This proteincontains 4 of the motifs.

DNA topoisomerase II alpha (Topo II α), which functions inpost-replicative DNA decatenation and DNA segregation. This proteincontains one motif.

the key nucleotide excision repair protein XPA which recognizes helicalkinks. This protein contains one motif.

RAD51B, a homologous recombination protein which shown to be importantfor proper centrosome function and chromosome segregation. This proteincontains one motif.

Fanconi anemia core complex protein, FANCC. The FA core complex is shownto be involved in the DNA damage-activated signalling pathway regulatingDNA repair of crosslinking agents. This protein contains one motif.

Further proteins which have been found to contain at least one motif arelisted in Table 1.

Without wishing to be bound by theory, the inventors findings indicatethat by preventing PCNA from interacting with at least one of its usualpartners, cells may be sensitised to the effect of cytostatic agents.Thus, the effect of the cytostatic agent may be modulated. For example,interaction of a repair protein with PCNA may be inhibited (e.g. hABH2)thereby inhibiting DNA repair, and as a consequence increasing theeffect of the cytostatic agent in damaging the DNA.

Thus, in a further aspect, there is provided a method of treating adisorder or condition, particularly a disorder or condition where it isdesirable to inhibit the growth of cells, for example ahyperproliferative disorder, or any condition which requires or isresponsive to cytostatic therapy, said method comprising administering(particularly administering an effective amount of) an oligopeptidiccompound as defined herein, a nucleic acid molecule as defined hereinand/or a vector as defined herein, to a subject in need thereof.

In another aspect, there is provided an oligopeptidic compound asdefined herein, a nucleic acid molecule as defined herein and/or avector as defined herein, for use in therapy, particularly for use inthe treatment of a disorder or condition where it is desirable toinhibit the growth of cells, for example a hyperproliferative disorder,or in any treatment which involves cytostatic therapy (i.e. the use of acytostatic agent). Thus the compound etc. may be used in the treatmentof any condition which requires or is responsive to cytostatic therapy.

In another aspect, there is provided the use of an oligopeptidiccompound as defined herein, a nucleic acid molecule as defined hereinand/or a vector as defined herein, in the manufacture of a medicamentfor use in the treatment of a disorder or condition where it isdesirable to inhibit the growth of cells, for example ahyperproliferative disorder, or in a treatment which involves cytostatictherapy.

As noted above, one surprising finding leading up to this invention isthat the effect of a range of different cytostatic drugs may be enhancedor potentiated by the use of a peptide having a PCNA-interacting motif,thereby to inhibit the interaction of PCNA with a presumably broad rangeof proteins, for example proteins involved in DNA repair and replicationand cell cycle progression etc. This leads to the general proposal thatany PCNA-interacting molecule may be used in combination with acytostatic agent, in order to enhance the effect of that cytostaticagent, or to sensitise cells to its effect.

Accordingly, in yet another aspect, there is provided a method oftreating a disorder or condition where it is desirable to inhibit thegrowth of cells, for example a hyperproliferative disorder, or a methodof treatment which involves cytostatic therapy, said method comprisingadministration of an oligopeptidic compound capable of interacting withPCNA or a nucleic acid molecule which comprises a nucleotide sequencethat encodes an oligopeptidic compound capable of interacting with PCNA,and separate, simultaneous or sequential administration of a cytostaticagent to a subject in need thereof.

Alternatively viewed, there is provided an oligopeptidic compoundcapable of interacting with PCNA or a nucleic acid molecule comprising anucleotide sequence which encodes said oligopeptidic compound for use incombination with a cytostatic agent in the treatment of a disorder orcondition where it is desirable to inhibit the growth of cells, forexample a hyperproliferative disorder, or in a treatment which involvescytostatic therapy.

Thus, there is provided the use of an oligopeptidic compound capable ofinteracting with PCNA or a nucleic acid molecule comprising a nucleotidesequence which encodes said oligopeptidic compound in the manufacture ofa medicament for use in combination with a cytostatic agent in thetreatment of a disorder or condition where it is desirable to inhibitthe growth of cells, for example a hyperproliferative disorder, or in atreatment which involves cytostatic therapy.

Thus, in one embodiment the medicament may further comprise a cytostaticagent.

The medicament may be in the form of a single composition comprisingboth the oligopeptidic compound or nucleic acid molecule and thecytostatic agent, or it may be in the form of a kit or productcontaining them for separate (e.g. simultaneous or sequential)administration.

There is thus also provided the use of an oligopeptidic compound capableof interacting with PCNA or a nucleic acid molecule comprising anucleotide sequence which encodes said oligopeptidic compound in themanufacture of a medicament for the treatment of a disorder of cells,for example a hyperproliferative disorder, or in a treatment whichinvolves cytostatic therapy, wherein the medicament is administeredseparately, simultaneously or sequentially with a cytostatic agent.

In another aspect, the invention provides a product containing anoligopeptidic compound capable of interacting with PCNA or a nucleicacid molecule comprising a nucleotide sequence which encodes saidoligopeptidic compound together with a cytostatic agent as a combinedpreparation for separate, simultaneous or sequential use in thetreatment of a disorder or condition where it is desirable to inhibitthe growth of cells, for example a hyperproliferative disorder, or in atreatment which involves cytostatic therapy.

The oligopeptidic compound capable of interacting with PCNA, preferablythe oligopeptidic compound comprising or having SEQ ID NO: 1, may beused to modulate or potentiate the effect of a cytostatic agent.

The oligopeptidic compounds (including constructs) according to theinvention thus have a therapeutic utility in any condition or clinicalsituation where it is desirable (or where it may be of benefit) toinhibit the growth of cells.

The term “inhibit” is used broadly to include any reduction or decreasein cell growth as well as the prevention or abolition of cell growth.“Inhibition” thus includes the reduction or prevention of cell growth.This may be determined by any appropriate or convenient means, such asdetermining or assessing cell number, size (e.g size of tissue in whichthe cells are contained), cell viability and/or cell death etc., as maybe determined by techniques well known in the art.

“Growth” of cells as referred to herein is also used broadly to includeany aspect of cell growth, including in particular the proliferation ofcells.

The oligopeptidic compounds may thus be used in the treatment of anycondition (used broadly herein to include any disorder or any clinicalsituation) which is responsive to reduction of cell growth (particularlycell proliferation). The oligopeptidic compounds accordingly findutility in any therapy (or treatment) which targets cell growth (orproliferation). In other words, the compounds may be used in anytherapeutic application in which it desirable or advantageous to inhibitcell proliferation.

The term “treatment” as used herein refers broadly to any effect or step(or intervention) beneficial in the management of a clinical conditionand thus includes both therapeutic and prophylactic treatments.Treatment may include reducing, alleviating, ameliorating, slowing thedevelopment of, or eliminating the condition or one or more symptomsthereof, which is being treated, relative to the condition or symptomprior to the treatment, or in any way improving the clinical status ofthe subject. A treatment may include any clinical step or interventionwhich contributes to, or is a part of, a treatment programme or regimen.A prophylactic treatment may include delaying, limiting, reducing orpreventing the condition or the onset of the condition, or one or moresymptoms thereof, for example relative to the condition or symptom priorto the prophylactic treatment. Prophylaxis thus explicitly includes bothabsolute prevention of occurrence or development of the condition, orsymptom thereof, and any delay in the onset or development of thecondition or symptom, or reduction or limitation on the development orprogression of the condition or symptom. Treatment according to theinvention thus includes killing, inhibiting or slowing the growth ofcells, or the increase in size of a body or population of cells (e.g ina tissue, tumour or growth), reducing cell number or preventing spreadof cells (e.g to another anatomic site), reducing the size of a cellgrowth etc. The term “treatment” does not imply cure or completeabolition or elimination of cell growth, or a growth of cells.

Since the therapeutic applications and utilities of the presentinvention may generally involve inhibiting cell proliferation, anyproliferating cell may be targeted in the therapies and utilitiesdisclosed and encompassed herein. Such proliferating cells may includehealthy or diseased cells and cells of any tissue in which proliferationoccurs. For example, such cells may include in particular neoplasticcells, including both malignant and non-malignant neoplastic cells andcells of the immune system (immune cells), cells of the haematopoieticsystem generally, or skin cells.

Disorders or conditions involving abnormal or unwanted cell growth maybe treated with cytostatic agents, and cytostatic agents may be used inany situation where it is desired to reduce or prevent cell growth andproliferation, including situations where it is desired to kill orablate cells. Accordingly, as alternatively stated above, theoligopeptidic compounds (including constructs) of the present inventionmay be used in any method of treatment which involves (or includes) theuse of a cytostatic agent. This may include the treatment of anycondition responsive to a cytostatic agent or any condition which may betreated with or which requires the use of a cytostatic agent.

The treatment of hyperproliferative disorders represents an aspect ofparticular interest. The term “hyperproliferative disorder” is usedbroadly herein to include any disorder or condition which involvesincreased, undesired or unwanted proliferation of cells. Thus includedare not only conditions in which proliferation of cells is increased,for example relative to normal or healthy cells, or cells in the absenceof the condition in question (e.g. compared or relative to a healthy orcontrol subject, or compared or relative to cells taken from healthy orunaffected tissue in the same subject), but also conditions in whichcell proliferation is not increased (or not greatly or significantlyincreased) over normal, but in which the proliferation which occurs isunwanted or undesired, whether generally or in a particular context.This may include for example an unwanted or undesired proliferation ofcells which may occur in a “normal” response, e.g. an immune response oran inflammatory response etc. (in other words a “normal” response whichmay occur in a particular (e.g. normal) context, but which maynonetheless be unwanted). Such an unwanted proliferative response mayfor example be the proliferation of cells resulting in an unwantedinflammatory response, or an unwanted immune response such as anautoimmune response or an allergic reaction etc.

Hyperproliferative disorders which may be treated according to thepresent invention thus explicitly include inflammation (moreparticularly inflammatory disorders or conditions, or conditionsinvolving, or associated with, or characterised by, inflammation) andautoimmune disorders or conditions, or disorders or conditions whichhave an autoimmune component.

A hyperproliferative disorder may involve (but is not limited to) theproliferation of cells which have the capacity for autonomous growthi.e. cells which exist and reproduce independently of normal regulatorymechanisms. A hyperproliferative disorder may therefore be a neoplasticdisorder, and as noted above, this may be a malignant or non-malignantdisorder.

Hyperproliferative cells may be classified as pathological (i.e.deviating from normal cells and associated with a disease state) ornon-pathological (i.e. deviating from normal but associated with adisease state).

Pathological hyperproliferative cells may be associated with, orcharacteristic of the following disease states or disorders: restenosis,diabetic nephropathy, thyroid hyperplasia, Grave's Disease, psoriasis,benign prostatic hypertrophy, Li-Fraumeni syndrome, and cancers(including any tumours or malignancies).

Examples of non-pathological hyperproliferative cells include mammaryductal epithelial cells during development of lactation and also cellsassociated with wound repair.

The compounds of the invention may be useful in the treatment of suchdisorders and diseases and others, including diabetic retinopathy andperipheral vascular diseases.

Hyperproliferative disorders may as noted above be malignant ornon-malignant neoplastic disorders. Also included are pre-malignant andnon-neoplastic disorders. Examples of pre-malignant or non-neoplastic ornon-malignant hyperproliferative disorders include myelodysplasticdisorders, cervical carcinoma-in-situ, familial intestinal polyposes(e.g. Gardner syndrome), oral leukoplakias, histiocytoses, keloids,hemangiomas, hyperproliferative arterial stenosis, inflammatoryarthritis, hyperkeratoses, and papulosquamous eruptions, includingarthritis. Also included are viral-induced hyperproliferative diseasessuch as warts and EBV-induced disease (e.g. infectious mononucleosis),scar formation and the like.

The hyperproliferative disorder may thus be any hyperproliferativedisorder, for example selected from neoplastic disorders such as cancer,psoriatic arthritis, rheumatoid arthritis, gastric hyperproliferativedisorders such as inflammatory bowel disease, skin disorders includingpsoriasis, Reiter's syndrome, pityriasis rubra pilaris, andhyperproliferative variants of the disorders of keratinization.

Cancer represents a hyperproliferative disorder of particular interest,and all types of cancers, including e.g. solid tumours andhaematological cancers are included. Representative types of cancerinclude cervical cancer, uterine cancer, ovarian cancer, pancreaticcancer, kidney cancer, gallbladder cancer, liver cancer, head and neckcancer, squamous cell carcinoma, gastrointestinal cancer, breast cancer,prostate cancer, testicular cancer, lung cancer, non-small cell lungcancer, non-Hodgkin's lymphoma, multiple myeloma, leukemia (such asacute lymphocytic leukemia, chronic lymphocytic leukemia, acutemyelogenous leukemia, and chronic myelogenous leukemia), brain cancer(e.g. astrocytoma, glioblastoma, medulloblastoma), neuroblastoma,sarcomas, colon cancer, rectum cancer, stomach cancer, anal cancer,bladder cancer, pancreatic cancer, endometrial cancer, plasmacytoma,lymphomas, retinoblastoma, Wilm's tumor, Ewing sarcoma, melanoma andother skin cancers.

Mention may be made also of sinus tumours, urethral and genito-urinarycancers, oesophageal cancer, myeloma, endocrine cancers, osteosarcoma,angiosarcoma, and fibrosarcoma, and any tumour of the peripheral orcentral nervous systems, malignant or benign, including gliomas andneuroblastomas.

Autoimmune disorders or diseases represent a further condition ofparticular interest, and include for example rheumatoid arthritis,multiple sclerosis, immune disorders such as systemic lupuserythematosus (SLE; lupus) or myasthenia gravis.

Also of interest, generally speaking, are haematological disorders, ordiseases of the blood or bone marrow, which need not necessarily bemalignant or cancerous (e.g various dyscrasias, or dysplasias,non-malignant hyperplasis, granuloma or MGUS (Monoclonal Gammopathy ofUnknown Significance). Thus, any condition which involves an unwanted,or undesired or abnormal proliferation of blood or bone marrow cells, ortheir precursors, may be treated according to the present invention.

Other conditions which may be particularly mentioned include neoplasticmeningitis and myeloproliferative diseases e.g. polycythemia vera (whichoccurs when excessive red blood cells are produced).

Various conditions may also occur as a result of, or may be otherwiseassociated with, inflammation or with an autoimmune disease. Suchconditions may also be treated according to the present invention.Particular mention may be made of scleromyxedema and papular mucinosis,amyloidosis and Wegener's granulomatosis.

As noted above, the compounds of the invention may augment or potentiatethe effects of a cytostatic agent. Accordingly, they may find utility inany therapeutic application where a cytostatic agent may be used. Thismay include any situation where it is desired to kill or ablate cells,which may include not only diseased cells. In particular, such asituation arises where is it desirable to ablate bone marrow prior totransplantation. The compounds of the invention may thus be used inmyeloablation, and particularly myeloablation preceding a transplant,which may for example be a bone marrow transplant, or more generally ahaemopoietic stem cell transplant (HSCT), (as well as from bone marrow,haemopoietic stem cells may also be obtained or derived from blood, e.g.peripheral blood).

Stem cell transplantation may be used in the treatment of diseases orconditions of the blood or bone marrow (i.e. haematological conditionsor disorders), which may be malignant or non-malignant, and certainother types of cancer, including solid tumour cancers such asneuroblastoma, Desmoplastic small round cell cancer, Ewing's sarcoma andchoriocarcinoma. Haematological malignancies include leukaemias,lymphomas (Hodgkin's and non-Hodgkin's) and myelomas. Non-malignanthaematological disorders include phagocyte disorders (e.g.myelodysplasia), anaemias (e.g. severe aplasia or aplastic anaemia), andmyeloproliferative disorders (e.g. polycythemia vera and essentialthrombocytosis). Other acquired conditions which may be treated by stemcell transplantation include metabolic disorders such as amyloidosis,and environmentally-induced diseases such as radiation poisoning. Stemcell transplantation may also be used in the treatment of congenitaldisorders, including various lysosomal storage disorders,immunodeficiencies and non-malignant haematological disorders e.g.anaemias, cytopenias, haemophagocytic syndromes, haemoglobinopathies,sickle cell disease and β thalassemia major.

Radiotherapy (also known as radiation therapy and radiation oncology)may be used in the treatment of various conditions including thehyperproliferative disorders described above. By “radiotherapy” is meantthe use of ionizing radiation which is capable of damaging the DNA ofcells by directly or indirectly ionizing the atoms which make up the DNAchain. Indirect ionization happens as a result of the ionization ofwater, forming free radicals, notably hydroxyl radicals, which thendamage the DNA. In the most common forms of radiotherapy, most of theradiation effect is through free radicals.

Radiotherapy is useful in the treatment of cancer and for ablation ofpathologic tissues because of the cytotoxic effects which result frompersistent DNA double strand breaks or activation of programmed celldeath. Ionizing radiation causes hyperproliferating cells, such astumour and cancer cells, to undergo cell death by apoptosis, both invivo and in vitro.

Unfortunately radiotherapy is often unsuccessful at completelyeradicating cancer cells from a patient because it is often not possibleto deliver a sufficiently high dose of local radiation to kill tumourcells without an unacceptably high risk of damage to the surroundingnormal tissue. It is also known that cells show widely varyingsusceptibilities to radiation-induced cell death and ionizing radiationcan also activate a pro-survival response mechanism throughphosphatidylinositol 3-kinase/Akt (PI3K/Akt) and mitogen-activatedprotein kinase (MAPK) signal transduction pathways. Thus, there is aneed to enhance the efficacy of radiotherapy by sensitizing cells to theeffects of ionizing radiation.

Accordingly, the compounds of the invention may be used to provide sucha sensitizing effect, in other words to enhance (or alternatively put toincrease, augment, or potentiate) the effects of radiotherapy, or torender a subject (or more particularly cells, which may be present in asubject) more susceptible to the effects of radiotherapy. Thus, they mayfind utility in any therapeutic application where radiotherapy is used.This may include any situation where it is desired to kill or ablatecells, which may include not only diseased cells.

The compounds of the invention may thus be used as a sensitizer of cellsto the DNA damaging effects of ionizing radiation. By “sensitizer” ismeant the use of the compounds of the invention to enhance the DNAdamaging effect of ionizing radiation on cells. This may be achieved bythe inhibition of the endogenous cellular DNA repair mechanisms.

Thus, the present invention encompasses an oligopeptidic compoundcomprising a PCNA interacting motif (more specifically an oligopeptidiccompound comprising a PCNA interacting motif as defined herein), or anucleic acid molecule comprising a sequence encoding said PCNAinteracting motif, for use in combination with radiotherapy, wherein thecompound is administered separately, simultaneously or sequentially withthe radiotherapy. The radiotherapy, together with the compound, may beadministered in the treatment of any condition which is responsive to,or which requires, radiotherapy. The compounds or constructs of theinvention may thus be used in the treatment of a disorder or conditionwhere it is desirable to inhibit growth of cells, for example ahyperproliferative disorder, or in any treatment which involvesradiotherapy,

Alternatively defined, the invention provides an oligopeptidic compoundcomprising a PCNA-interacting motif (more specifically an oligopeptidiccompound comprising a PCNA interacting motif as defined herein) or anucleic acid molecule comprising a sequence encoding said PCNAinteracting motif, as a sensitizer for radiotherapy, wherein thecompound is administered separately, simultaneously or sequentially withradiotherapy.

These aspects of the invention also provide a method of sensitizing asubject (or more particularly cells or tissue in said subject) toradiotherapy, which method comprises administering to said subject anoligopeptidic compound of the invention as defined herein, particularlyan amount of said compound which is effective to sensitize said subject(or said cells or tissue) to the radiotherapy.

This aspect of the invention can also be seen to provide a method oftreating a subject, said method comprising administering radiotherapy tosaid subject, in conjunction with an oligopeptidic compound of theinvention as defined herein. More particularly such a method may be amethod of treatment of a disorder or condition which is responsive to,or which requires, radiotherapy, or a disorder or condition in which itis desirable to inhibit the growth of cells, or a method of treatmentwhich involves radiotherapy.

The invention contemplates all types of radiotherapy including, but notlimited to Conventional external beam radiotherapy, StereotacticRadiotherapy, Virtual simulation, 3-dimensional conformal radiotherapy,intensity-modulated radiotherapy and Radioisotope Therapy (RIT).

Thus in one preferred embodiment of any of the aspects listed herein,the oligopeptidic compound, nucleic acid molecule and/or vector asdefined herein is/are used in conjunction (simultaneously, separately orsequentially) with a radiotherapy.

In a further preferred embodiment of any of the aspects listed herein,the oligopeptidic compound, nucleic acid molecule and/or vector asdefined herein is/are used in conjunction (simultaneously, separately orsequentially) with a cytostatic agent.

By “cytostatic agent” is meant an agent which is capable of inhibitingor suppressing the growth and/or multiplication(replication/proliferation) of animal cells.

Included as cytostatic agents are cytotoxic agents, anti-neoplasticagents and any agent which may be indicated for an oncological orhaematological application. Thus, included are agents used inchemotherapeutic treatment protocols (“chemotherapeutic agents”).

Cytostatic agents are typically grouped into different classes accordingto their mechanism of action and all of these classes are contemplatedherein. Thus, the cytostatic agent may be an alkylating agent, across-linking agent, an intercalating agent, a nucleotide analogue, aninhibitor of spindle formation, and/or an inhibitor of topoisomerase Iand/or II. Other types or classes of agent include anti-metabolites,plant alkaloids and terpenoids, or an anti-tumour antibiotic.Preferably, it is an alkylating agent.

Alkylating agents modify DNA by alkylating nucleosides, which leads tothe prevention of correct DNA replication. Nucleotide analogues becomeincorporated into DNA during replication and inhibit DNA synthesis.Inhibitors of spindle formation disturb spindle formation, leading tothe arrest of mitosis during metaphase. Intercalating agents intercalatebetween DNA bases, thereby inhibiting DNA synthesis. Inhibitors oftopoisomerase I or II affect the torsion of DNA, thereby interferingwith DNA replication.

Suitable cytostatic agents are known in the art, but by way of exampleactinomycin D, BCNU (carmustine), carboplatin, CCNU, Camptothecin (CPT),cantharidin, Cisplatin, cyclophosphamide, cytarabine, dacarbazine,daunorubicin, docetaxel, Doxorubicin, DTIC, epirubicin, Etoposide,gefinitib, gemcitabine, ifosfamide irinotecan, ionomycin, Melphalan,Methotrexate, Mitomycin C (MMC), mitoxantronemercaptopurine,Oxaliplatin, Paclitaxel (taxol), PARP-1 inhibitor, taxotere,temozolomide (TZM), teniposide, topotecan, treosulfan vinorelbine,vincristine, vinblastine, 5-Azacytidine, 5,6-Dihydro-5-azacytidine and5-fluorouracil are named herein. The skilled person will be aware ofsuitable dosage ranges for any given cytostatic agent and in oneembodiment, the cytostatic agent is present in the pharmaceuticalcomposition, or administered to the subject, in its typical dose range.In an advantageous embodiment, a lower dose of the cytostatic agent maybe present/used, because the oligopeptidic compound, nucleic acidmolecule or vector of the invention sensitises the cells to thecytostatic agents and so when used in combination with the oligopeptidiccompound, nucleic acid molecule or vector of the invention, a lower doseof the cytostatic agent will have the same or a comparable therapeuticeffect as a higher dose of the cytostatic agent on its own. Theoligopeptidic compound, nucleic acid molecule or vector of the inventiontherefore makes it possible to treat subjects which have a low, or lowerthan average, tolerance for cytostatic agents, such as old people,babies or young children, or people weakened e.g. through disease,malnutrition and the like.

One problem encountered when using cytostatic agents to treat ahyperproliferative disorder is that typically not all of the affectedcells are killed. It is envisaged that when an cytostatic agent and theoligopeptidic compound, nucleic acid molecule or vector of the inventionare used together, a higher percentage of affected cells are killed andin one embodiment, a higher than typical dose of the cytostatic agent isused together with the oligopeptidic compound, nucleic acid molecule orvector of the invention to achieve killing of a very high proportion ofthe diseased cells, e.g. at least 50, 60 70 or 80%, preferably at least85, 90 or 95%, most preferably to kill substantially all of the diseasedcells.

As noted above, when the oligopeptidic compound, nucleic acid moleculeand/or vector as defined herein is used in conjunction with a cytostaticagent, then the two different agents may be present in the samepharmaceutical composition, or they may be administered separately.Separate administration may include administration at substantially thesame time but via different routes of administration or byadministration at different locations. Separate administration may alsoinclude administration at different times, e.g. up to 1, 2, 3, 4, 5, 6or 12 hours apart.

The subject is an animal (i.e. any human or non-human animal),preferably a mammal, most preferably a human.

The skilled person will be well aware of suitable methods forintroducing the oligopeptidic compound, nucleic acid molecule and/orvector into cells. By way of example, a few suitable methods are brieflydiscussed below. As discussed in detail above, peptide-mediated methodsof delivery can be used, notably cell penetrating peptides (CPPs), whichas discussed above, are short, in some cases polycationic, sequenceswhich can facilitate cellular uptake of peptides, proteins or nucleotidemolecules which contain CPPs or to which CPPs are linked, for example byenhancing uptake into endosomes of mammalian cells. Microencapsulationprovides a simple and cost-effective way to enclose bioactive materialswithin a semi-permeable polymeric membrane for the purpose of protectingthe bioactive materials and releasing the enclosed substances or theirproducts in a controlled fashion. In photochemical internalisation (PCI)both the molecule of interest and a photosensitising compound are takenup by the cell into a lysosome or an endosome. The cells are thenexposed to light of suitable wavelengths to activate thephotosensitising compound, causing the photosensitising compound todisrupt the membrane of the lysosome or endosome, thereby releasing themolecule of interest into the cytosol of the cell.

Other methods include microinjection, red blood cell ghost-mediatedfusion, liposome fusion, osmotic lysis of pinosomes, scrape loading,electroporation, calcium phosphate and virus-mediated transfection andthe use of copolymeric carriers.

Chitosan and water-soluble chitosan derivatives, in particular glycolchitosan, are emerging as the drug carriers of choice because of theirbiocompatibility and biodegradability in vivo. A preferred example isglycol chitosan hydrophobically modified with 5 β-cholanic acid.

The standard amino acid one letter code is used herein, so K stands forlysine (Lys), I stands for isoleucine (Ile) and so on.

The oligopeptidic compound of the invention may incorporate one or more,e.g. at least 1, 2, 3, 4 or 5 amino acids which possess a side chainthat is not coded for by the standard genetic code, termed herein“non-coded amino acids”. These may be selected from amino acids whichare formed through metabolic processes such as ornithine or taurine,and/or artificially modified amino acids such as9H-fluoren-9-ylmethoxycarbonyl (Fmoc), (tert)-(B)utyl (o)xy (c)arbonyl(Boc), 2,2,5,7,8-pentamethylchroman-6-sulphonyl (Pmc) protected aminoacids, or amino acids having the benzyloxy-carbonyl (Z) group.Preferably, where such non-coded amino acids are present, they are notlocated within the motif, but in one embodiment one or more non-codedamino acids are present within the motif.

In vitro and/or in vivo stability of the oligopeptidic compound of theinvention may be improved or enhanced through the use of stabilising orprotecting means known in the art, for example the addition ofprotecting or stabilising groups, incorporation of amino acidderivatives or analogues or chemical modification of amino acids, Suchprotecting or stabilising groups may for example be added at the Nand/or C-terminus. An example of such a group is an acetyl group andother protecting groups or groups which might stabilise a peptide areknown in the art.

The oligopeptidic compounds of the invention will typically compriseonly amino acids having the L-configuration, but one or more amino acidshaving the D configuration may be present. Preferably, the oligopeptidiccompound contains at least 1, 2, 3, 4 or 5 D-amino acids and they arepreferably found in the motif, but in another embodiment, D amino acidsare present only outside of the motif. The oligopeptidic compound may belinear or cyclic.

Thus, included particularly are inverso oligopeptidic compounds orinverso analogues of the oligopeptidic compounds of the invention (andmore particularly inverso peptides).

Also included are retro oligopeptidic compounds (or retro peptides) inwhich the residues (e.g. amino acid residues) are assembled in oppositedirection to the parental or reference compound (e.g. peptide).

Retro-inverso oligopeptidic compounds include D-amino acids in reverse(opposite) order to the parental or reference compound sequence. Aretro-inverso analogue thus has reversed termini and reversed order ofe.g. peptide bonds, while approximately maintaining the topology of theside chains as in the parental or reference sequence.

The compounds of the invention may include partial inverso, retro orretro-inverso sequences.

By “oligopeptidic compound” is meant a compound which is composed ofamino acids or equivalent subunits, which are linked together by peptideor equivalent bonds. Thus, the term “oligopeptidic compound” includespeptides and peptidomimetics.

By “equivalent subunit” is meant a subunit which is structurally andfunctionally similar to an amino acid. The backbone moiety of thesubunit may differ from a standard amino acid, e.g. it may incorporateone or more nitrogen atoms instead of one or more carbon atoms.

By “peptidomimetic” is meant a compound which is functionally equivalentor similar to a peptide and which can adopt a three-dimensionalstructure similar to its peptide counterparts, but which is not solelycomposed of amino acids linked by peptide bonds. A preferred class ofpeptidomimetics are peptoids, i.e. N-substituted glycines. Peptoids areclosely related to their natural peptide counterparts, but they differchemically in that their side chains are appended to nitrogen atomsalong the molecule's backbone, rather than to the α-carbons as they arein amino acids.

In a preferred embodiment, at least the motif part of the oligopeptidiccompound comprises only peptide bonds and preferably it is composedsolely of coded amino acids. Most preferably, the oligopeptidic compoundis a peptide.

The oligopeptidic compound may incorporate di-amino acids and/or β-aminoacids, but at least the motif part is preferably only composed ofα-amino acids. Most preferably, the oligopeptidic compound consists ofα-amino acids.

The prefix “oligo” is used to designate a relatively small number ofsubunits such as amino acids, i.e. less than 200, preferably less than100, 90, 80, 70 60 or 50 subunits. The oligopeptidic compound of theinvention may thus comprise at least 5 and no more than 200 subunits.Preferably, it comprises at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19 or 20 subunits. Alternatively defined it comprises nomore than 40, 35, 30, 29, 28, 27, 26 or 25 subunits. Representativesubunit ranges thus include 5-40, 5-35, 5-30, 5-25, 5-20, 5-15, 5-12,5-10 etc, 5-20 and 5-30 being preferred.

When the oligopeptidic compound comprises more than 5 subunits, then thenature of the subunits outside of the motif is not critical, so thesubunits outside of the motif may for example be those found in thenative protein such as hABH2, or they may be alanine residues or anyother suitable residues.

Peptidomimetics typically have a longer half life within a patient'sbody, so they are preferred in embodiments where a longer lasting effectis desired. This can help reduce the frequency at which the compositionhas to be re-administered. However, for bio-safety reasons a shorterhalf life may be preferred in other embodiments; in those embodimentspeptides are preferred.

The oligopeptidic compound of the invention may form part of a largerunit, e.g. it may be fused to a polypeptide to form a recombinant fusionprotein or attached to a scaffold to form a peptide aptamer. Thus,fusions proteins or aptamers incorporating the oligopeptidic compound ofthe invention form further aspects of the present invention. Yet furtheraspects include pharmaceutical compositions comprising such fusionsproteins or aptamers and the use of such fusions proteins or aptamers intherapy or in a method of treatment as described above.

Without wishing to be bound by theory, it is believed that for optimumDNA repair, maintenance and/or cell cycle regulation, several proteinshave to interact with PCNA and that the oligopeptidic compounds of theinvention are able to compete with proteins which possess the consensusmotif for interacting with PCNA. Examples of proteins which may have tointeract with PCNA via the novel motif for optimum DNA repair,maintenance and/or cell cycle regulation are listed in Table 1, so theprotein may be a DNA polymerase, DNA ligase, Topoisomerase, DNA repairprotein, DNA repair associated/interacting proteins, a protein involvedin Sister chromatid cohesion, Chromatin remodelling, DNA binding,Ubiquitin processing or SUMO processing, E3 ubiquitin ligase,Transcription factor, Cell cycle regulator, Protein kinase,Methyltransferase, Acetyl-transferase, Cancer associated antigen,Structural protein or Centrosome kinesin.

When a sufficient level of the oligopeptidic compound of the inventionis present within a cell, then the activity of one or more of theseproteins is reduced or even abolished due to this competitiveinhibition.

The oligopeptidic compound, nucleic acid molecule or vector as definedherein on its own is believed to have no enzymatic activity and to benon-toxic to cells (see Example 2). Thus, it has been shown thatexpression of a peptide of the invention comprising a PCNA-interactingmotif as defined herein has no effect, or only minor effects, on thegrowth of the cell in which it is expressed. This may be dependent onthe expression level. However, in some situations it is believed thatthe oligopeptidic compounds of the invention may be cytotoxic andaccordingly they may be used as cytotoxic (or cytostatic) agents intheir own right. Thus, the compounds of the invention may be used ascytotoxic agents in the treatment of conditions as discussed herein, andnot necessarily always in combination with a separate cytostatic agent,or with radiotherapy.

Experiments have shown that oligopeptidic compounds administered tocells may have a cytotoxic effect on the cell.

The cytotoxic effect may vary depending on the precise nature of thecompound, for example its sequence or composition. In particular, thecytotoxic effect may be obtained with constructs comprising an NLSand/or CPP and has been observed with constructs containing both and NLSand CPP. Strong evidence of cytotoxicity has been observed withnuclear-localising constructs.

Oligopeptidic compounds which exhibit a cytotoxic effect may be inverso,retro or retro-inverso etc.

More particularly, it has further been observed that an increasedcytotoxic effect may be obtained with compound or constructs whichcontain more than one PCNA-binding motif according to the presentinvention.

In a further aspect, there is provided herein a kit, or a pharmaceuticalproduct, comprising

(i) an oligopeptidic compound as defined herein, a nucleic acid moleculeas defined herein, and/or a vector as defined herein; and

(ii) a cytostatic agent.

In another aspect there is provided a product containing (i) anoligopeptidic compound as defined herein, a nucleic acid molecule asdefined herein, and/or a vector as defined herein and (ii) a cytostaticagent as a combined preparation for simultaneous, sequential or separateuse in the treatment of a disorder or condition where it is desirable toinhibit the growth of cells, for example a hyperproliferative disorder,or in a treatment which involves cytostatic therapy.

Also contemplated is the in vitro administration of an oligopeptidiccompound, nucleic acid molecule and/or a vector as defined herein to acell or cell culture. Such in vitro methods may be used to study DNArepair, maintenance and/or cell cycle regulation. In a preferred aspect,the in vitro method is used to identify novel cytostatic agents. Thismay allow the faster identification of cytostatic agents, or theidentification of agents which are only weakly cytostatic when used ontheir own, but which have useful cytostatic activity when used incombination with the oligopeptidic compound, nucleic acid molecule orvector of the invention.

The novel PCNA interacting motif as defined herein may be used in thediagnosis or monitoring of a disorder or condition where it is desirableto inhibit the growth of cells, for example a hyperproliferativedisorder, or a treatment which involves cytostatic therapy orradiotherapy.

The inventors have found that several cancer associated antigens possessthe PCNA-binding motif (see Table 1). It is therefore envisaged that ahyperproliferative or other disorder as discussed above may be diagnosedor its progress may be monitored by detecting the level of expressionand/or location of a protein containing the motif, wherein an aberrantlevel and/or location of the protein is indicative of the disorder, e.g.a hyperproliferative disorder.

By an “aberrant level” is meant an increased level of the protein, e.g.the level is more than 10, 20, 30 or 40% compared to the level in ahealthy cell of the same cell type, or a decreased level of the protein,e.g. the level is less than 10, 20, 30 or 40% compared to the level in ahealthy cell of the same cell type.

In a preferred embodiment, an increased level of a protein containingthe motif is indicative of a disorder, e.g. hyperproliferative disorder.

The level of the motif-containing protein can be analyzed using anyknown protein detection method. Preferably, an antibody specific for themotif is used. The antibody must be sufficiently specific for the motif(as compared to a reference protein such as Bovine serum albumin) for itto be used in a diagnostic method.

The antibody can be a monoclonal or a polyclonal antibody and it may bea whole antibody, e.g. IgG, IgA, IgE, IgM, or IgD, or an antibodyfragment such as Fab, Fab′, F(ab′)₂, scFv, Fv, dsFv, ds-scFv, Fd, dAbs.

The detection assay may be carried out in vivo or in vitro, e.g. in atissue or cell or body fluid sample, e.g. a cellular lysate, serum orblood.

Detection can be facilitated by coupling (i.e., physically linking) theantibody to a detectable substance (i.e., antibody labeling). Examplesof detectable substances include various enzymes, prosthetic groups,fluorescent materials, luminescent materials, bioluminescent materials,NMR contrast agents and radioactive materials. Examples of suitableenzymes include horseradish peroxidase, alkaline phosphatase,luciferase, beta-galactosidase, acetylcholinesterase, glucose oxidase,lysozyme, malate dehydrogenase and the like; examples of suitableprosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes luminol; examples ofbioluminescent materials include luciferase, luciferin, and aequorin,and examples of suitable radioactive material include ¹²⁵I, ¹³¹I, ³⁵S or³H. In the case of a direct visual label, use may be made of a colloidalmetallic or non-metallic particle.

Preferably there is provided a method of diagnosing or monitoring theprogress in a subject of a disorder or condition in which it isdesirable to inhibit the growth of cells, for example ahyperproliferative disorder,

said method said method comprising the steps of:

-   -   (1) contacting a test sample taken from said subject (e.g.        mammal) with an antibody specific for the motif under conditions        that permit the formulation of an antibody-antigen complex;    -   (2) measuring the amount of antibody-antigen complex in the test        sample; and    -   (3) comparing the amount of antibody-antigen complex in the test        sample to a control.        The control may be a healthy cell taken from the same subject,        e.g. a healthy fibroblast cell.

In another aspect, the invention relates to antibodies specific for themotif.

The invention further includes a kit for diagnosing or monitoring adisorder or condition where it is desirable to inhibit the growth ofcells, for example a hyperproliferative disorder,

said kit comprising an antibody specific for the motif and instructionsfor the use thereof to diagnose the disorder or condition. Preferably,the antibody is coupled to a detectable substance as described above, orthe kit includes such a detectable substance.

An alternative method of diagnosis involves the detection of an aberrantlevel of a nucleic acid molecule which encodes the motif. In thismethod, the level of the nucleic acid is monitored using a suitabledetection method such as the polymerase chain reaction (PCR) orhybridisation techniques using suitably labeled probes.

EXAMPLES Experimental Procedures Used in the Examples ExpressionConstructs.

Cloning of the fluorescently tagged expression constructs ECFP-PCNA andhABH2₁₋₂₆₁-EYFP has previously been described (Aas et al., 2003).Employing hABH2₁₋₂₆₁-EYFP as a template, hABH2₁₋₁₀-EYFP andhABH2₁₁₋₂₆₁-EYFP were generated by PCR. The amplicons were cloned intopEYFP-N1 (Clontech) using NdeI/AgeI and AgeI/EcoRI respectively. The PCRproduct from EST (Image clone 5176979 (BC035374) RZPD) was cloned intopEYFP-C1 (HindIII/Acc651) to give EYFP-TFIIS-L. The EYFP-XPA constructwas made by switching EYFP with EGFP (NheI/BsrGI fragment) inHis9-HA-EGFP-XPA (Rademakers et al., 2003) generously provided by Dr.Wim Vermeulen (Department of Cell Biology and Genetics, Rotterdam).TFII-I-EYFP was generated by PCR amplification of TFII-I frompI3CX-TFII-I (Roy et al., 1993) generously provided by Dr. Robert G.Roeder (Laboratory of Biochemistry and Molecular Biology, TheRockefeller University, New York) and cloning into EYFP-N1 (SacI/ApaI).EYFP-Topo-IIa was made by switching the EGFP tag (EcoRI blunt/NheI) withthe EYFP tag (XhoI blunt/NheI) from EGFP-Topo-IIa (pT104-1)(Mo and Beck,1999) generously provided by William T. Beck (Division of MolecularPharmacology, Department of Molecular Genetics, University of Illinois,Chicago). The hABH2₁₋₇-EYFP constructs including the F4 mutants, weremade by annealing oligos with XhoI/EcoRI overhang followed by cloninginto EYFP-N1 mutated in the ATG codon. All point mutations were made bysite direct mutagenesis according to the QuickChange® II instructionmanual. Restriction enzymes and Calf Intestinal Alkaline Phosphatase(CIP) were from New England Biolabs® Inc. and the oligonucleotides werefrom MedProbe, Eurogentech (Oslo, Norway). All constructs were verifiedby sequencing.

Confocal Imaging and FRET Measurements

Live HeLa cells were examined 16-24 hours after transient transfection(by Fugene 6 (Roche Inc.) according to the manufacturer'srecommendations) of ECFP and EYFP fusion constructs. Fluorescent imageswere acquired using a Zeiss LSM 510 Meta laser scanning microscopeequipped with a Plan-Apochromat 63×/1.4 oil immersion objective.Enhanced cyan fluorescent protein (ECFP) was excited at λ=458 nm anddetected at λ=470-500 nm and enhanced yellow fluorescent protein (EYFP)was excited at λ=514 nm and detected at λ=530-600 nm, using consecutivescans. The thickness of the slice was 1 μm.

Fluorescent resonance energy transfer (FRET) occurs if the tags (EYFPand ECFP) are less than 100 Å (10 nm) apart. We detected FRET using thesensitised emission method, measuring acceptor (EYFP) emission upondonor (ECFP) excitation. We had FRET when the intensity of emitted lightfrom EYFP after excitation of the ECFP fluorochrome was stronger thanthe light emitted by ECFP or EYFP-tagged proteins alone, afterexcitation with the EYFP and ECFP lasers respectively (bleed through),given by the equation: FRET=I₂−I₁ (I_(D2)/I_(D1))−I₃ (I_(A2)/I_(A3))is >0. FRET was normalised for expression levels using the equation:N_(FRET)=FRET/(I₁×I₃)^(1/2). N_(FRET) was calculated from meanintensities (I) within a region of interest (ROI) containing more than25 pixels where all pixels had intensities below 250 and the averageintensities were between 100 and 200 for both the donor and the acceptorconstructs. Channel 1 (ECFP) and 3 (EYFP) were measured as describedabove for imaging, and channel 2 (FRET) was excitated with λ=458 nm anddetected at λ=530-600 nm. I_(D1, D2, D3) and I_(A1, A2, A3) weredetermined for cells transfected with ECFP and EYFP constructs only,with same settings and same fluorescence intensities as co-transfectedcells (I₁ and I₃). ECFP-PCNA and EYFP-PCNA were included as positivecontrols, and due to dimerisation of co-expressed tags, ECFP and EYFPproteins expressed from empty vectors were included as negative controlsin all experiments.

Culture of Cell Lines and Preparation of Cell Extracts

HeLa (cervical cancer) and HaCaT (spontaneously transformedkeratinocyte) cells stably expressing the constructs of interest wereprepared by transfection (by Fugene 6) followed by cell sorting orcloning by dilution, and prolonged culturing in selective (usinggeneticin, G418, 400 μg/ml, Invitrogen) Dulbecco's modified Eagle'sMedium high glucose 4.5 g/l(DMEM) (BioWhittaker®) supplemented with 10%Fetal Calf Serum (FCS), Amphotericin B (250 μg/ml, Sigma-Aldrich),gentamycin (100 μg/ml, Gibco) and glutamine (1 mM, BioWhittaker®). Thecells were cultured at 37° C. in 5% carbon dioxide, humidifiedatmosphere. Fractionated cell extracts from HeLa were prepared byresuspending the cell pellets in 1× packed cell volume (PCV) in buffer I(10 mM Tris-HCl, pH 8.0 and 50 mM KCl) and 1×PCV in buffer II (10 mMTris-HCl, 100 mM KCl, 20% glycerol, 0.5% Nonidet P-40, 10 mM EGTA, 10 mMMgCl₂, 1 mM DTT, 1× Complete protease inhibitor (Roche), phosphatesinhibitor cocktail (PIC I and PIC II, Sigma). Cells were incubated underconstant shaking for 30 min at 4° C. The supernatant (soluble fraction)was harvested. The pellet (containing nuclei) was resuspended in 1×PCVof buffer III (10 mM Tris-HCl, pH 8.0 and 100 mM KCl) and 1×PCV bufferII, and briefly sonicated until all nuclei were disrupted. 750 μg of thenucleoli-containing fraction was centrifuged and the pellet (chromatinbound fraction) was resuspended in buffer II and III. The chromatinbound fraction was incubated with DNAse/RNAse cocktail (2 μl Omnicleave®Endonuclease (200 U/μl, Epicentre® Biotechnologies, WI), 2 μl DNAse (10U/μl, Roche Inc.), 2 μl Benzonase (250 U/μl, Novagen, Ge), 2 μlMicrococcal Nuclease (100-300 U/μl, Sigma-Aldrich) and 2 μl RNAse (10mg/ml, Sigma-Aldrich) for 30 min at room temperature and 1 h at 37° C.750 μg of the soluble fraction was incubated with additional 2 μlOmnicleave® over night at 4° C. during IP.

Co-Immunoprecipitation (Co-IP) and Western Analysis (WB).

An in-house affinity purified rabbit polyclonal antibody raised againstGFP protein, which also recognises EYFP and ECFP proteins, wascovalently linked to protein-A paramagnetic beads (Dynal®) according toprocedure from New England Biolabs® Inc (from now on called α-GFPbeads). Each fraction was incubated with α-GFP beads (10 μl) duringconstant rotation at 4° C. over night (IP). After IP, the beads werewashed 4 times with 200 μl 10 mM Tris-HCl, 50 mM KCl (pH 7.5), with 5min incubation on ice in between. The beads were then resuspended inNuPAGE® (Invitrogen) loading buffer and 1 mM DTT, heated, and separatedon 10% or 4-12% Bis-Tris-HCl(NuPAGE®) gels and transferred to PVDFmembranes (Immobilon®, Millipore). The membranes were blocked for 1 h in5% low fat dry milk in PBST (PBS with 0.1% Tween® 20). The primaryantibodies, α-PCNA (PC10, Santa Cruz biotechnology Inc.) and α-GFP werediluted in 1% dry milk in PBST and incubated for 1 h, followed by 1 hincubation with secondary antibodies, Polyclonal Rabbit Anti-mouseIgG/HRP and Polyclonal Swine Anti-rabbit IgG/HRP, respectively(DakoCytomation, Denmark). The membranes were treated withchemiluminescence reagent (SuperSignal® West Femto Maximum, PIERCE), andthe proteins visualised in Kodak Image Station 2000R.

Sequence Analysis

For initial sequence analysis the Swiss-Prot and TrEMBL databases wereused to find proteins with similar sub-sequences as the sequence regionof interest. The databases were queried with motifs in PROSITE format.Clustal W was used to align the sequences of interest. The conservedmotifs listed in Supplementary File 1 were identified by comparison ofgene orthologs. Data files for Inparanoid version 5.1 were downloadedfrom the Inparanoid web server <http://inparanoid.sbc.su.se/> for arepresentative subset of organisms. The human sequences were used asreference, and the Inparanoid processed fasta file was searched with aregular expression for the APIM motif, using a local tool. A slightlyexpanded motif definition was used, where Ala was allowed at eitherposition 3 or 4 of the motif, in addition to Ile, Val and Leu, but notat both positions simultaneously. From a total of 22218 proteinsequences there were 636 sequences with at least one hit against theAPIM motif. These entries were matched against experimental andpredicted subcellular localisation in the eSLDB database, downloadedfrom the web server <http://gper.biocomp.unibo.it/esldb/>, and 349entries with no indication of targeting to the nucleus were removed. Forthe remaining 287 entries the corresponding Inparanoid orthologs wereidentified, the corresponding sequences were extracted from the fastafiles, and the resulting sequence libraries were aligned with Clustal W.

The 24 sequence entries without orthologs in Inparanoid were removedfrom the analysis. Two different procedures were used in parallel foridentification of conserved sites. In the first procedure (Consensus)the consensus sequence was estimated from the multiple alignment foreach hit position in the human sequence. When estimating the consensusequivalent symbols in the conservative APIM motif (without Ala) wheretreated as equivalent symbols for estimation of the consensus, so thate.g. Ile, Val and Leu were treated as a single residue type. The regularexpression was tested again against the consensus before the hitposition was accepted. In the alternative procedure (Individual) theregular expression was tested against each orthologous subsequencecorresponding to a hit position in the human sequence, and onlypositions where at least 50% of the orthologs matched the expressionwere accepted. In this estimate subsequences consisting only of gapswere excluded, assuming that this could represent e.g. alternativesplice variants. These two procedures gave almost identical results, andthe combined output is shown in Supplementary File 1. In total 37entries were removed by this procedure, the remaining 226 entries werelisted and analysed. The protein descriptions used in the output weretaken from the Inparanoid unprocessed human fasta file and Ensemblrelease 45. The output file is in html format and can be opened by astandard web browser.

Dot-Blot Analysis of Predicted PCNA-Binding Peptides

An Amino-PEG500-UC540 sheet (acid hardened with improved stability)containing dots of 28 nmol peptide (dyed with Ponceau to visualise thespots) was prepared at the peptide synthesis lab at The Biotechnologycentre at University of Oslo, Norway. The membrane was probed with 1μg/ml PCNA for 2 h, followed by probing with primary antibody (α-PCNA,PC10) and developed as described above for WB.

Cell Survival Assay

HeLa and HaCaT cells were seeded into 96 well plates (4000 cells/well)and incubated for 4 hours. Various doses of MMS (methylmethanesulfonate, Acros), BCNU (1,3-Bis(2-chloroethyl)-1-nitrosourea,Sigma), temozolomide (4-methyl-5-oxo-2,3,4,6,8-pentazabicyclo [4.3.0]nona-2,7,9-triene-9-carboxamide, TZM, Sigma) and mitomycin C(6-Amino-1,1a,2,8,8a,8b-hexahydro-8-(hydroxymethyl)-8a-methoxy-5-methyl-azirino[2′,3′:3,4] pyrrolo[1,2-a]indole-4,7-dione carbamate, MMC, Sigma) were addedto the wells. The U2OS cells were exposed to MMS and TMZ only. The cellswere exposed continuously until harvest. Cells were harvested every dayfor 4 days using the MTT assay (Mosmann, 1983). OD was measured at 570nm, and the average from at least 6 wells was used to calculate cellsurvival. Data presented is growth from one representative experimentand has been reproduced at least 2 times.

Example 1

This work described in this Example investigates the localisation ofhABH2 in replication foci and identifies direct interaction betweenhABH2 and PCNA, and the region of hABH2 responsible for such aninteraction.

In living S-phase cells, PCNA tagged with green fluorescent protein(EGFP) forms distinct foci representing sites of replication and thuscan be used as a S-phase marker.

PCNA tagged with cyan fluorescent protein (ECFP) was co-expressed withvarious hABH2 deletion constructs fused with yellow fluorescent protein(EYFP). It was found that deletion of the 10 N-terminal amino acids inhABH2 (hABH2₁₁₋₂₆₁-EYFP) totally abolished the co-localisation with PCNAin replication foci. Remarkably, when these 10 amino acids were fused toEYFP (to give a construct called hABH2₁₋₁₀-EYFP), they were sufficientfor co-localisation with PCNA. (FIG. 1). Notably, co-expression ofECFP-PCNA increased the localisation of full length hABH2(hABH2₁₋₂₆₁-EYFP), as well as hABH2₁₋₁₀-EYFP, in nuclear foci, comparedto cells expressing hABH2 constructs alone. This suggests a directinteraction between PCNA and hABH2 mediated by the 10 N-terminal aminoacids of hABH2.

To examine the degree of proximity of hABH2 and PCNA, fluorescenceresonance energy transfer (FRET) was measured. Both full lengthhABH2-EYFP and hABH2₁₋₁₀-EYFP generated positive FRET with ECFP-PCNA,demonstrating that the distance between the fluorescent tags is lessthan 100 Å. This suggests that the hABH2 variants directly interact withor are in the same complex as ECFP-PCNA (FIG. 2).

To confirm a direct interaction, co-immunoprecipitation studies wereperformed using protein extracts from cells stably expressinghABH2-EYFP, hABH2₁₁₋₂₆₁-EYFP, hABH2₁₋₁₀-EYFP or EYFP.Anti-GFP-antibodies were used to immunoprecipitate the respective fusionproteins. Subsequent western blot analyses revealed that the endogenousPCNA was pulled down by hABH2₁₋₂₁₆-EYFP and hABH2₁₋₁₀-EYFP, but not byhABH2₁₁₋₂₆₁-EYFP or EYFP. Taken together, these results suggest thathABH2 directly interacts with PCNA and that the binding sequence iscontained within hABH2s 10 N-terminal amino acids.

Example 2

The ability of hABH2₁₋₁₀ to inhibit hABH2 was tested.

Cell lines expressing hABH2₁₋₁₀-EYFP or EYFP alone were exposed to thealkylating agents MMS (methyl methanesulfonate), BCNU (Carmustine),temozolomide (TZM) or mitomycin C (MMC). MMS is an S_(N)2 alkylatingagent that leads to 3-methylcytosine (3meC) and 1-methyladenine (1meA)which are repaired by hABH2, while BCNU is an O⁶-chloroethylating agentthat mainly leads to interstrand crosslinks as well as some mono-basecyclic adducts (1,N(6)ethenoadenine). TZM is reported to be an O⁶Gmethylating agent, while MMC causes interstrand cross links viaN-alkylation of guanine in CpG's.

Over-expression of hABH2₁₋₁₀-EYFP or EYFP did not interfere with thegrowth rate of untreated cells; however it was found that expression ofhABH2₁₋₁₀-EYFP sensitised HeLa cells to treatment with MMS.Surprisingly, it was also found that expression of hABH2₁₋₁₀-EYFPsensitised HeLa cells to all of the other cytostatic agents tested (FIG.3). This indicates that the increased sensitivity was not only caused byinhibition of hABH2, which is believed mainly to repair 3meC and 1meA.

It is known from a previous study that mouse Abh2^(−/−) cells, i.e. ABH2knock-out cells, display increased sensitivity to MMS, but not to BCNU.Therefore, the increased sensitivity of cells expressing hABH2₁₋₁₀-EYFPto cytostatic agents such as BCNU cannot be explained solely byinhibition of hABH2.

HaCaT (spontaneously transformed keratinocyte) cells stably expressinghABH2₁₋₁₀-EYFP were also hypersensitive to MMS and TMZ.

Flow and comet assay analysis of the cells after treatment with thedifferent alkylating agents showed that the drugs affected the cellcycle differently, and induced different levels and patterns of DNArepair intermediates (abasic sites, SSB, DSB) as detected by the cometassay. MMS lead to an S-phase arrest (day 1) and gave the highest levelsof DNA repair intermediates after 4 hours. However, MMS treated cellsshowed normal cell cycle distribution and no increased levels of DNArepair intermediates on day 2. Similar to MMS, TMZ also lead to thehighest levels of repair intermediates after 4 hours, however the cellswere arrested in G2/M up to day 3 indicating a different damage andrepair pattern. TMZ also induced more strand breaks than all the otherthree agents tested. This is surprising since TMZ is believed to inducemainly O⁶methyl-G which is repair by a direct repair mechanism by MGMT,not involving removal of any bases or strand breaks. BCNU treatment leadto en transient G2/M arrest on day 1 and very low levels of repairintermediates, indicating that most cells containing crosslinks died.MMC treatment on the other hand, lead to an S-phase arrest on day 1 and2, and this was more pronounced for the hABH2₁₋₁₀-EYFP expressing cellsthan for the control cells. The cells were still arrested in G2/M on day3. No significant differences between the cells lines could be observedin the comet assay after MMC treatment, but the number of repairintermediates topped at day 2.

Generally, it was not possible to detect any significant differences inthe amount of repair intermediates between the EYFP and hABH2₁₋₁₀-EYFPexpressing cells by the comet assay. The comet and flow analysis showthat the different agents used induces both different cell cycleresponses and different patterns of repair. Still all agents hadincreased cytotoxicity in APIM expressing cells compared to EYFPexpressing cells, thus supporting a role for several APIM containingproteins in the regulation between repair or cell death.

Example 3

An alignment of database sequences ABH2s from several different speciesrevealed that the 7 N-terminal amino acids are highly conserved (FIG.4). To identify a binding sequence, the importance of these amino acidsfor the peptide-PCNA interaction was examined using a dot blot assay. Itwas inter alia found that Arg3 and Lys7 could substitute each other andthat Leu5 and Val6 could be substituted by each other or by otheraliphatic amino acids such as Ile and Ala without affecting the apparentaffinity towards PCNA. Furthermore, the completely conserved aromaticamino acid, Phe4 could be replaced by Tyr, whereas Ala in this positionsignificantly reduced the PCNA binding.

Substitutions of amino acids 1-2 and 8-10 with Ala did not affect PCNAbinding, suggesting the pentapeptide RFLVK (SEQ ID NO. 2) as the coreinteracting sequence.

Further assays revealed that this pentapeptide was by itself sufficientfor PCNA binding, but additional flanking amino acids increased theinteraction.

Next, amino acids 1-7 of hABH2, and variants of this sequence in whichPhe4 was replaced by Tyr, Trp or Ala, were expressed in fusion with EYFPand tested for co-localisation with PCNA in vivo. Similar to what wasfound in the dot blot assay, fusion proteins containing an aromaticamino acid in position 4 co-localised with ECFP-PCNA, while proteinswith Ala in this position did not.

Example 4

The Swiss-Prot and TrEMBL databases were used to find proteins withsimilar sub-sequences as the sequence which had been identified as beingresponsible for binding of hABH2 to PCNA. Using the consensus[KR]-[FYW]-[LIVA]-[LIVA]-[KR] (SEQ ID NO. 30) as the query, 226 hitswere obtained (see Table 1 for a summary), of which several humanproteins were chosen for further analysis.

One was a protein which like hABH2 contains the above consensus sequencewithin its 7 N-terminal amino acids. This protein also contains theconserved N-terminal domain I found in TFIIS elongation factor, animportant protein for progression of stalled transcription. Theinventors named this protein TFIIS-like protein (TFIIS-L). The functionof this protein is unknown.

The second protein was the multifunctional transcription factor TFII-I,which contains 4 consensus sequences. TFII-I is critical for cell cyclecontrol and proliferation, and cells over-expressing TFII-I haveincreased persistence of γ-H2AX foci (a marker for DNA double strandbreaks), suggesting a role for TFII-I in DNA repair.

DNA topoisomerase II alpha (Topo II α) containing one consensus sequencewas also examined. Topo II α functions in post-replicative DNAdecatenation and DNA segregation.

A consensus sequence was also found internally in the key nucleotideexcision repair protein (NER) XPA which recognizes helical kinks.

A consensus sequence was found in RAD51B, a homologous recombinationprotein which has been shown to be important for proper centrosomefunction and chromosome segregation.

Another protein was the Fanconi anemia core complex protein, FANCC,which was found to contain one consensus sequence. Fanconi anemia (FA)is a rare genetic disorder characterized by aplastic anemia, increasedleukaemia susceptibility and hypersensitivity against crosslinkingagents. The FA core complex has been shown to be involved in the DNAdamage-activated signalling pathway regulating DNA repair ofcrosslinking agents.

In all these proteins the putative PCNA_binding motif was found to beare conserved across different species (FIG. 5). Among these fiveproteins, only Topo II α and FANCC (after damage) has been reported toco-localise with BRCA1 in nuclear S-phase foci and Topo II α is the onlyprotein containing a potential PIP-box (QttLaFkp, aa 1277-84). Theinventors now showed that EYFP fusion proteins of each and every ofthese proteins co-localise with ECFP-PCNA in S-phase foci.

Other interesting APIM containing proteins found are members of thePoly(ADP-ribose) family (PARP-1, 2 and 4) involved in several DNAmaintenance processes including DNA repair, the PARP-1 partner and TopoII α isoform DNA topoisomerase II beta involved in resolution oftopological problems caused by replication forks. Furthermore, APIM isfound in the REV3L subunit of the translesion polymerase ζ, involved inboth point mutagenesis and larger scale genome stability, the DNA ligaseI and IV involved in both DNA replication and repair, the four E3ubiquitin-protein ligases (UHRF1 and UHRF2/NIRF, UBR1 and 2), allinvolved in regulation of the cell cycle, genome maintenance andintegrity, as well as several other E3 ubiquitin ligases (Table 1).Interestingly, E3 ubiquitin ligase are frequently shown to begenetically and expressionally altered in breast tumorigenesis. Alsocontaining APIM is the N-acetyltransferase ESCO1/EFO1, a protein whichyeast orthologue bind PCNA through its truncated PIP-box and which isinvolved in proper sister chromatid cohesion, and the human structuralmaintenance of chromosome protein 5, hSMC5, which is shown to beinvolved in DNA repair of double strand breaks through HR and inmaintenance of telomeres in ALT cells. Finally, several subunits of thegeneral transcription factors II and III, subunits of RNA polymerase IIand serin/threonine protein kinases are found to contain the APIM motif(Table 1).

TABLE 1 Type/group of proteins Proteins containing APIM DNA polymerasePol zeta catalytic subunit (hREV3L)¹ DNA ligase DNA ligase I¹, DNAligase IV Topoisomerase Topo II alpha and Topo II beta² DNA repairprotein hABH2*, XPA*, PARP-1³, 2 and 4, RAD51B*, FANCC*⁴ DNA repairassociated/ XPA-binding protein 2, BRCA1/BRCA2- interacting proteinscontaining complex subunit 45 (prot-BRE), X-ray radiation resistanceassociated protein 1 Sister chromatid cohesion N-acetyltransferaseESCO1/EFO1¹, hSMC5⁵ Chromatin remodelling Chromodomainhelicase-DNA-binding protein 3, and DNA binding 4 and 5, p325 subunit ofRSF chromatin proteins remodelling complex, Telomeric repeat-bindingprotein 2 (TRF2)⁶ E3 ubiquitin ligases UHFR1, UHFR2, UBR1, UBR2, Ringfinger proteins 3, 17 and 151, Probable E3 ubiquitin- protein ligaseMYCBP2 Ubiquitin processing Ubiquitin-specific-processing protease(FAF-X) SUMO processing Sentrin/SUMO-specific protease SENP2Transcription factors TFIIS-L*, TFII-I*, TFIIE-alpha, Sterol regulatoryelement binding transcription factor 2(SREBF2), TFIIIC subunit alpha,TFIID 100 kDa subunit (TAF5), TFIIIC 102 kDa subunit (TF3C gamma),Transcription factor-like protein MRG15 and X (Mortality factor 4-likeprotein 1 and 2), E2F transcription factor 7 Cell cycle regulators Celldivision cycle associated 2, Bcl2-interacting mediator of cell death,Testis spermatocyte apoptosis-related gene 2 protein Protein kinasesSerine/Threonine (S/T) -protein kinases SRPK1 and 2, 33 and MST4,Leucine-rich repeat S/T- protein kinase 1, STK23 (S/T protein kinase23), S/T protein kinase PLK3, Microtubuli-associated S/T-protein kinase,Microtubuli-associated S/T- protein kinase 1, P13-kinase p110 subunitgamma, Interferon-inducible double stranded RNA- dependent proteinkinase activator A, FYVE finger-containing phosphoinositide kinas,Phosphoinositide 3-Kinase-C2-beta, Phosphatidylinositol-4-phosphate5-kinase type II alpha and beta, Phosphatidylinositol-4,5- bisphosphate3-kinase catalytic subunit alpha isoform, MAPKAP kinase 2 and 5,Mitogen- activated protein kinase 15 (MAP 15) Methyltransferase H3lysine-4 specific MLL3, H3-K9 methyltransferase 5, Putative rRNAmethyltransferase 3 Acetyl-transferase Diacetylglycerol O-acetyltransferase (DGAT1) Cancer associated Melanoma associated antigen E1,MAGE E1, antigens MAGE B18, MAGE-G1, NK-tumor recognition protein(NK-TR), Myc-binding protein associated protein, Myb binding protein 1A,Hepatoma- derived growth factor related protein 2 isoform 1,Serologically defined colon cancer antigen 1 Structural proteinsLamin-B1 and B2, Actin-like protein 2 Centrosom, kinesins, Centrosomalprotein 110 kDA (Cep110), Centrosomal protein 192 kDa, Microtubule plusend directed kinesin motor 3 (KIF3A), Kinesin heavy chain (UKHC),kinetochore-associated protein 1 Bold: proteins localised in replicationfoci under normal conditions or after DNA damage. This study* orelsewhere: ¹ G. L. Moldovan, B. Pfander, and S. Jentsch, Cell 129 (4),665 (2007). ² Z. Lou, K. Minter-Dykhouse, and J. Chen, Nat Struct MolBiol 12 (7), 589 (2005); A. Niimi, N. Suka, M. Harata et al., Chromosoma110 (2), 102 (2001). ³ C. M. Simbulan-Rosenthal, D. S. Rosenthal, S.Iyer et al., Molecular and cellular biochemistry 193 (1-2), 137 (1999).⁴ C. Jacquemont and T. Taniguchi, BMC biochemistry 8 Suppl 1, S10(2007). ⁵ P. R. Potts, M. H. Porteus, and H. Yu, Embo J 25 (14), 3377(2006). ⁶ P. L. Opresko, M. Otterlei, J. Graakjaer et al., Mol Cell 14(6), 763 (2004).

Example 5

The function of the consensus sequence in the proteins studied inExample 4 was experimentally examined. Because substitution of thearomatic amino acid Phe in the consensus sequence with Ala abolishedPCNA interaction in vitro and co-localisation with PCNA in vivo, it wasexamined whether the corresponding mutation had similar effect on thefull-length proteins. Mutation of Phe4 to Ala in full length hABH2abolished co-localisation with PCNA. In TFII-I, containing 4 of themotifs, the Phe residues (F431A, F536A, F641A and F803A) weresubstituted individually and collectively to Ala. No single mutationreduced co-localisation with PCNA, but mutations in all of the consensussequences in TFII-I strongly reduced the co-localisation with PCNA inreplication foci, showing that several consensus sequence motifs presentin the same protein may contribute to optimal PCNA interaction.

REFERENCES

-   Aas, P. A., Otterlei, M., Falnes, P. O., Vagbo, C. B., Skorpen, F.,    Akbari, M., Sundheim, O., Bjoras, M., Slupphaug, G., Seeberg, E.,    and Krokan, H. E. (2003). Human and bacterial oxidative demethylases    repair alkylation damage in both RNA and DNA. Nature 421, 859-863.-   Mo, Y. Y., and Beck, W. T. (1999). Association of human DNA    topoisomerase IIalpha with mitotic chromosomes in mammalian cells is    independent of its catalytic activity. Experimental cell research    252, 50-62.-   Mosmann, T. (1983). Rapid colorimetric assay for cellular growth and    survival: application to proliferation and cytotoxicity assays.    Journal of immunological methods 65, 55-63.-   Rademakers, S., Volker, M., Hoogstraten, D., Nigg, A. L., Mone, M.    J., Van Zeeland, A. A., Hoeijmakers, J. H., Houtsmuller, A. B., and    Vermeulen, W. (2003). Xeroderma pigmentosum group A protein loads as    a separate factor onto DNA lesions. Mol Cell Biol 23, 5755-5767.-   Roy, A. L., Malik, S., Meisterernst, M., and Roeder, R. G. (1993).    An alternative pathway for transcription initiation involving    TFII-I. Nature 365, 355-359.

Example 6 Preparation and Testing of Constructs Containing a Motif(“APIM”)-Containing Peptide with Signal Sequences

Peptides of SEQ ID NOS. 98 to 117 were synthesized using standardtechniques, and fluorescent tags incorporated, where indicated, againusing standard techniques. The peptides are shown in Table 3, whichshows the amino acid sequence for each peptide, and also listsseparately the individual components making up each peptide (motif(“APIM”)-containing peptide, NLS, CPP, and linkers, as appropriate, andthe tag used, where it is contained) Peptides of SEQ ID NOS. 98 to 116are made up of L-amino acids. Peptide RI-MDR26-3 of SEQ ID NO. 117 is aretro-inverso peptide made up of D-amino acids. All of the peptidesshown in Table 3 have at least one APIM peptide, an NLS and a CPP.

Various studies were undertaken to show the effect of the peptides oncells. Thus cells were incubated with the peptides to determinelocalisation of the peptides in the cells, using techniques based onthose described in the Examples above. Briefly, the peptides indicatedto be labelled with fluorescent tags were incubated with the cells (HeLacells) and cellular import was examined using confocal microscopy.

The effects of the peptides in sensitising cells to the effects ofcytostatic drugs were investigated using an MTT assay and a clonogenicassay (CFU assay) as described below.

Cytotoxicity of the peptides was investigated by an MTT assay asdescribed below, using the peptides alone, in the absence of cytostaticdrugs. Cytotoxicity data was also obtained from controls used in thecytostatic drug MTT assays (controls with peptide but without cytostaticdrug), where the control peptides were followed for longer (4 days).

Membrane toxicity was investigated, again as described below.

Stability of the peptides was investigated by MS analysis of thepeptides after incubation of the peptides in serum-containing medium.

MTT Assay

HeLa cells were seeded into 96 well plates (6000 cells/well) andincubated for 3 hours. Various doses of MMS and Cisplatin were added tothe wells. After 24 hours peptides were added to the cells in serum freemedia and incubated for 1 h. Fresh media with cytostatic drugs was addedand the cells were harvested after additional 24, 48 and 72 hours. MTTwas added to the cells(3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and ODwas measured at 570 nm [52], and the average from at least 6 wells wasused to calculate cell survival. Data is presented as growth from onerepresentative experiment and has been reproduced at least 2 times.

Clonogenic (CFU) Assay

750 HeLa cells were seeded out in 10 cm cell culture dishes in 11 mlgrowth media containing cytostatic drugs (MMS). The 2^(nd) day the cellswere treated with peptides for one hour under serum free conditions.Fresh media containing fresh cytostatic drugs were added to the cellsfor further incubation for 10 days. The cells were then fixed in 6%glutaraldehyde in PBS for 15 min at room temperature, washed once in PBSand stained with crystal violet and colony forming units were counted.Only colonies consisting of at least 50 cells were included.

Cytotoxicity Assay

Cytotoxicity of the peptides after 48 hours are measured using the MTTassay. HeLa cells were seeded into 96 well plates (6000 cells/well) andincubated for 3 hours. Various doses of peptides were added to the wellsin presence or absence of serum in the media. After 1 h equal volumemedia with 1× or 2× (to serum free media) were added and the cellsincubated for 48 hours before addition of MTT (see above).

Membrane Toxicity by Flow Cytometry

The cells were treated with different concentrations (2, 4 and 8 μM) ofpeptide for 1 hour. Next, propidium iodide (PI) (50 μg/ml in PBS) wasadded, this will stain DNA if the cell membranes are permeable. Analysiswere done within the next 10 minutes on a FACS Canto flow cytometer(BD-Life Science).

The results are shown in Table 4. From this it can be seen that alltag-labelled peptides tested, which are essentially constructs of anAPIM-containing peptide with a CPP and an NLS, localised to the nucleus.This effect was seen for peptides with different linker sequenceslinking the individual components of the construct, linkers of variablelength, and peptides without linker sequences. This shows that thepresence of linker sequences is not essential and the linker sequencecan be varied.

The results of the MTT and CFU experiments with cytostatic agents showthe effect of peptides in increasing the growth inhibitory effects ofthe cytostatic agents. Thus, the peptides are able to sensitise thecells to the effects of the cytostatic agents. This effect is analogousto that reported in Example 2 above for transfected cell-linesexpressing an APIM-containing peptide.

Table 4 also shows a cytotoxic effect of a number of peptides. Furtherexperimental work (data not shown) has indicated that a higher cytotoxiceffect is observed with peptides which localise to the nucleus, comparedwith those that do not. A membrane toxicity experiment carried out withone peptide (MDR2; SEQ ID NO. 98) appears to indicate that membranetoxicity is low, which may suggest that the cytotoxic effect observedfor the peptides is not due to a membrane effect. It has been observedthat in some cases the cytotoxic effect may be seen only with increasedpeptide concentrations (for example, at 104 peptide cytotoxicity may notbe seen (although an effect in sensitising cells to a cytostatic agentis seen), whereas at 2 μM peptide a cytotoxic effect of the peptideitself is seen, as well as the sensitising effect).

The results in Table 4 also show that the effects of the peptides inentering the cell and localising to the nucleus, in sensitising thecells to cytostatic agents, and in cytotoxicity may be obtained withdifferent NLS and/or CPP sequences and, whilst the magnitude or extentof the effect may vary, the effects do not appear to be dependent onparticular NLS and/or CPP sequences.

Certain peptides, as shown in Tables 3 and 4, incorporated an Ac groupat the N-terminus. This was included with the aim of stabilising thepeptides, both in serum and in the cytosol. Peptide MDR26-72-0 (SEQ IDNO. 112) showed good stability and good activity in the CFU and MTTassays, and the cytotoxicity tests. MDR26-72-0 contains an R-richsequence as a CPP. It is believed that equally good results would beobtained with equivalent peptides in which the CPP is replaced by a CPPderived or based on Penetratin or HIV-TAT, in which the NLS may be SV40or UNG2-derived).

TABLE3 SEQ ID NO: Peptide # Total Peptide Sequence APIM sequenceLinker 1 NLS Linker 2  98 MDR2MDRWLVKGAQPKKKRKVLRQIKIWFQNRRMKWKK-Ahx5-FAM)G MDRWLVK GAQ PKKKRKVL(SEQ ID NO: 118) (SEQ ID NO: 123)  99 MDR27MDRWLVKGAWKKKRVKIIRKKRRQRRRK-Ahx5-FAM)G MDRWLVK GAW KKKRVK II(SEQ ID NO: 118) (SEQ ID NO: 124) 100 MDR26-0MDRWLVKGAWKKKRKIIRKKRRQRRRG MDRWLVK GAW KKKRK II (SEQ ID NO: 118)(SEQ ID NO: 125) 101 MDR26-1 MDRWLVKGAWKKKRKIIRKKRRQRRRK-Ahx5-FAM)GMDRWLVK GAW KKKRK II (SEQ ID NO: 118) (SEQ ID NO: 125) 102 MDR26-2MDRWLVKRIWKKKRKIIRKKRRQRRRK-Ahx5-FAM)G MDRWLVK RIW KKKRK II(SEQ ID NO: 118) (SEQ ID NO: 125) 103 MDR26-3MDRWLVMANINKKKRKIIRKKRRQRRRK-Ahx5-FAM)G MDRWLVK WWW KKKRKII(SEQ ID NO: 118) (SEQ ID NO: 125) 104 MDR26-4MDRWLVKWWRKRHIIKKRKKRRQRRRK-Ahx5-FAM)G MDRWLVK WWW RKRHIIKK(SEQ ID NO: 118) (SEQ ID NO: 126) 105 MDR26-7MDRWLVKRIWKKKRKIIRRRRRRRRRRRK-Ahx5-FAM)G MDRWLVK RIW KKKRK II(SEQ ID NO: 118) (SEQ ID NO: 125) 106 MDR26-8MDRWLVKRIWKKKRKIIRQIKIWFQNRRMKWKK-Ahx5-FAM)G MDRFLVK RIW KKKRK II(SEQ ID NO: 119) (SEQ ID NO: 125) 107 MDR26-10MDRFLVKGAWRKRHIIKKRKKRRQRRRK-Ahx5-FAM)G MDRWLVK GAW RKRHIIKK(SEQ ID NO: 118) (SEQ ID NO: 126) 108 MDR26-72MDRWLVKWKKKRKIRRRRRRRRRRRK-FAM)G MDRWLVK W KKKRK I (SEQ ID NO: 118)(SEQ ID NO: 125) 109 MDR26-32 MDRWLVKWKKKRKIRKKRRQRRRK-FAM)G Ac-MDRWLVKW KKKRK I (SEQ ID NO: 120) (SEQ ID NO: 125) 110 MDR26-42MDRWLVKWRKRHIRKKRRQRRRK-FAM)G Ac-MDRALVK W RKRH I (SEQ ID NO: 121)(SEQ ID NO: 127) 111 MDR24-43 MDRWLVKGAWRKRHIRKKRRQRRRK-FITC Ac-MDRWLVKGAW RKRH I (SEQ ID NO: 120) (SEQ ID NO: 127) 112 MDR26-72-0Ac-MDRWLVKWWKKKRKIRRRRRRRRRRR MDRWLVK W KKKRK I (SEQ ID NO: 118)(SEQ ID NO: 125) 113 MDR26-72-A Ac-MDRALVKWKKKRKIRRRRRRRRRRR KVLWRDM WKKKRK I (SEQ ID NO: 122) (SEQ ID NO: 125) 114 MDR26-72-011Ac-MDRWLVKKKKRKRRRRRRRRRRRK-Ahx5-FAM)G MDRWLVK KKKRK (SEQ ID NO: 118)(SEQ ID NO: 125) 115 MDR26-72-01 Ac-MDRWLVKKKKRKRRRRRRRRRRR MDRWLVKKKKRK (SEQ ID NO: 118) (SEQ ID NO: 125) 116 MDR34MDRWLVKRRNKKKRKIIRWLVKWWWRKKRRQRRRK-Ahx5-FAM)G MDRWLVK RIW KKKRK II(SEQ ID NO: 118) (SEQ ID NO: 125) 117 RI-MDR26-3FITC-KRRRQRRKKRIIKRKKKWVWVKVLWRDM MDRWLVK WWW KRKKK II (SEQ ID NO: 118)(SEQ ID NO: 128)  98 MDR2 MDRWLVKGAQPKKKRKVLRQIKIWFQNRRMKWKK-Ahx5-FAM)GRQIKIWFQNRRMKWK K-Ahx-5-FAM)G (SEQ ID NO: 129)  99 MDR27MDRWLVKGAWKKKRVKIIRKKRRQRRRK-Ahx5-FAM)G RKKRRQRRR K-Ahx-5-FAM)G(SEQ ID NO: 130) 100 MDR26-0 MDRWLVKGAWKKKRKIIRKKRRQRRRG RKKRRQRRRGNotag (SEQ ID NO: 131) 101 MDR26-1MDRWLVKGAWKKKRKIIRKKRRQRRRK-Ahx5-FAM)G RKKRRQRRR K-Ahx-5-FAM)G(SEQ ID NO: 130) 102 MDR26-2 MDRWLVKRIWKKKRKIIRKKRRQRRRK-Ahx5-FAM)GRKKRRQRRR K-Ahx-5-FAM)G (SEQ ID NO: 130) 103 MDR26-3MDRWLVKWWWKKKRKIIRKKRRQRRRK-Ahx5-FAM)G RKKRRQRRR K-Ahx-5-FAM)G(SEQ ID NO: 130) 104 MDR26-4 MDRWLVKWWRKRHIIKKRKKRRQRRRK-Ahx5-FAM)GRKKRRQRRR K-Ahx-5-FAM)G (SEQ ID NO: 130) 105 MDR26-7MDRWLVKRIWKKKRKIIRRRRRRRRRRRK-Ahx5-FAM)G RRRRRRRRRRR K-Ahx-5-FAM)G(SEQ ID NO: 132) 106 MDR26-8MDRWLVKRIWKKKRKIIRQIKIWFQNRRMKWKK-Ahx5-FAM)G RQIKIWFQNRRMKWKK-Ahx-5-FAM)G (SEQ ID NO: 129) 107 MDR26-10MDRFLVKGAVVRKRHIIKKRKKRRQRRRK-Ahx5-FAM)G RKKRRQRRR K-Ahx-5-FAM)G(SEQ ID NO: 130) 108 MDR26-72 MDRWLVKWKKKRKIRRRRRRRRRRRK-FAM)GRRRRRRRRRRR K-FAMG (SEQ ID NO: 132) 109 MDR26-32MDRWLVKWKKKRKIRKKRRQRRRK-FAM)G RKKRRQRRR K-FAMG (SEQ ID NO: 130) 110MDR26-42 MDRWLVKWRKRHIRKKRRQRRRK-FAM)G RKKRRQRRR K-FAMG (SEQ ID NO: 130)111 MDR24-43 MDRWLVKGAWRKRHIRKKRRQRRRK-FITC RKKRRQRRR K-FITC(SEQ ID NO: 130) 112 MDR26-72-0 Ac-MDRWLVKWKKKRKIRRRRRRRRRRR RRRRRRRRRRRNotag (SEQ ID NO: 132) 113 MDR26-72-A Ac-MDRALVKWKKKRKIRRRRRRRRRRRRRRRRRRRRRR Notag (SEQ ID NO: 132) 114 MDR26-72-011Ac-MDRWLVKKKKRKRRRRRRRRRRRK-Ahx5-FAM)G RRRRRRRRRRR K-Ahx-5-FAM)G(SEQ ID NO: 132) 115 MDR26-72-01 Ac-MDRWLVKKKKRKRRRRRRRRRRR RRRRRRRRRRRNotag (SEQ ID NO: 132) 116 MDR34MDRWLVKRIWKKKRKIIRWLVKWWWRKKRRQRRRK-Ahx5-FAM)G RWLVK WWW RKKRRQRRRK-Ahx-5-FAM)G (SEQ ID NO: 134) (SEQ ID NO: 130) 117 RI-MDR26-3FITC-KRRRQRRKKRIIKRKKKWWWKVLWRDM RRRQRRKKR K-FITC (SEQ ID NO: 133)

TABLE4 SEQ ID Cyto- Membrane NO: Peptide # Total Peptide SequenceLocalization CFU MTT Stability toxicity Toxicity  98 MDR2MDRVVLVKGAQPKKKRKVLRQIKIWFQNRRMKWKK-Ahx5-FAM)G nucleus + +(+) Low  99MDR27 MDRWLVKGAWKKKRVKIIRKKRRQRRRK-Ahx5-FAM)G nucleus + + 100 MDR26-0MDRWLVKGAWKKKRKIIRKKRRQRRRG unknown +(+) + 101 MDR26-1MDRWLVKGAWKKKRKIIRKKRRQRRRK-Ahx5-FAM)G nucleus + + 102 MDR26-2MDRWLVKRIWKKKRKIIRKKRRQRRRK-Ahx5-FAM)G nucleus + + 103 MDR26-3MDRWLVKWWWKKKRKIIRKKRRQRRRK-Ahx5-FAM)G nucleus ++ ++ +(+) +(+) 104MDR26-4 MDRWLVKVWVRKRHIIKKRKKRRQRRRK-Ahx5-FAM)G nucleus ++ ++ +(+) +(+)105 MDR26-7 MDRWLVKRIWKKKRKIIRRRRRRRRRRRK-Ahx5-FAM)G nucleus ++ ++ ++(+) 106 MDR26-8 MDRWLVKRIWKKKRKIIRQIKIWFQNRRMKWKK-Ahx5-FAM)G nucleus ++++ ++ + 107 MDR26-10 MDRFLVKGAVVRKRHIIKKRKKRRQRRRK-Ahx5-FAM)G nucleus+(+) 108 MDR26-72 MDRWLVKWKKKRKIRRRRRRRRRRRK-FAM)G nucleus ++ ++ +(+)109 MDR26-32 MDRWLVKWKKKRKIRKKRRQRRRK-FAM)G nucleus ++ ++ +(+) 110MDR26-42 MDRWLVKWRKRHIRKKRRQRRRK-FAM)G nucleus ++ ++ +(+) 111 MDR24-43MDRWLVKGAWRKRHIRKKRRQRRRK-FITC nucleus ++ +(+) 112 MDR26-72-0Ac-MDRWLVKWKKKRKIRRRRRRRRRRR unknown +++ ++ ++ ++ 113 MDR26-72-AAc-MDRALVKWKKKRKIRRRRRRRRRRR unknown — (+) ++ (+) 114 MDR26-72-011Ac-MDRWLVKKKKRKRRRRRRRRRRRK-Ahx5-FAM)G nucleus — +(+) +++ (+) 115MDR26-72-01 Ac-MDRWLVKKKKRKRRRRRRRRRRR unknown — (+) +++ (+) 116 MDR34MDRWLVKRIWKKKRKIIRWLVKWWWRKKRRQRRRK-Ahx5-FAM)G nucleus — — ++ ++

Example 7 Further Cytotoxicity Data

In this Example more detailed data from cytotoxicity tests is presented.The cytotoxicity assay was performed as described in Example 6 above.Results are presented in FIG. 6 which shows the results of the MTTcytotoxicity assay for peptides MDR26-0 (SEQ ID NO. 100), MDR26-3 (SEQID NO. 103), MDR26-4 (SEQ ID NO. 104), MDR26-8 (SEQ ID NO. 106), MDR26-7(SEQ ID NO. 105), MDR26-72-0 (SEQ ID NO. 112), MDR26-72-01 (SEQ ID NO.115) and MDR34 (SEQ ID NO. 116).

It will be seen that cytotoxicity of the peptides can be observed, witha greater effect being seen in the absence of serum. Cytotoxicity isseen with all the MDR26 variant peptides, which have one APIM motif.Increased cytotoxicity is seen with MDR34 variants which have two APIMmotifs. Similar results showing cytotoxicity are obtained with peptidescorresponding to MDR34 (SEQ ID NO. 116) which have no tag (MDR34-0), orwhich are the inverso (I-MDR-34) or retroinverso (RI-MDR-34) equivalentsof MDR34. MDR34-2 which does not have an NLS sequence, and which insteadhad an extended linker sequence IILVIII (SEQ ID. NO. 95) as linker 2,shows reduced cytotoxicity.

Example 8

Further experiments have been done which support the interaction betweenPCNA and APIM-containing peptides., Co-immunoprecipitation (co-IP)experiments have showed that both endogenous PCNA and EYFP-PCNA fromcells stably expressing EYFP-PCNA were able to pull down hABH2. Moreinteraction seen between hABH2 and PCNA in chromatin-enriched fractionsindicates post-translational modifications on PCNA or hABH2. In theseexperiments co-IP of hABH2 from cells stably expressing EYFP-PCNA wasdemonstrated using magnetic beads coupled with antibodies againstα-EYFP. The membrane was probed with α-hABH2 and re-probed with α-PCNAantibody. Co-IP was also demonstrated of hABH2 from cells onlyexpressing endogenous proteins using magnetic beads coupled withantibodies against α-PCNA. The membrane was probed with α-hABH2 andreprobed with α-PCNA antibody.

Further experiments showing in vivo cross-linking support direct bindingbetween APIM and PCNA. Crosslinked and reversed crosslinked FLAG fusionproteins from cells stably expressing hABH21-7-EYFP 3×FLAG andhABH21-7-F4A-EYFP 3×FLAG were immunoprecipitated using α-FLAG AffinityGel. The IP elution fractions were analyzed by Western Blots usingα-PCNA or α-FLAG antibodies.

What is claimed is:
 1. A method of treatment of a disorder or conditionwhere it is desirable to inhibit the growth of cells, or a method oftreatment which involves cytostatic therapy, said method comprisingadministering an oligopeptidic compound to a subject in need thereof,wherein said oligopeptidic compound is capable of interacting withproliferating cell nuclear antigen (PCNA), wherein the compoundcomprises a PCNA interacting motif which is: (SEQ ID NO. 28)[K/R]-[F/Y/W]-[L/I/V/A/M]-[L/I/V/A/M]-[K/R];

wherein the oligopeptidic compound has 20-50 amino acids and comprises acell penetrating signal sequence selected from the group consisting ofSEQ ID NOs: 38-74, 129, 131 and 133, and wherein in said compound a PCNAinteracting motif is N-terminal to said cell penetrating signalsequence.
 2. The method of claim 1, wherein the oligopeptidic compoundfurther comprises a nuclear localization signal sequence.
 3. The methodof claim 1, wherein the PCNA interacting motif is: (SEQ ID NO. 29)[K/R]-[Y/W]-[L/I/V/A/M]-[L/I/V/A/M]-[K/R]; (SEQ ID NO. 30)[K/R]-[F/Y/W]-[L/I/V/A]-[L/I/V/A]-[K/R]; (SEQ ID NO. 31)[K/R]-[Y/W]-[L/I/V/A]-[L/I/V/A]-[K/R]; (SEQ ID NO. 32)[K/R]-[F/W]-[L/I/V/A/M]-[L/I/V/A/M]-[K/R]; (SEQ ID NO. 33)[K/R]-[F/W]-[L/I/V/A]-[L/I/V/A]-[K/R]; (SEQ ID NO. 34)[K/R]-[F/W]-[L/I/V]-[L/I/V]-[K/R]; (SEQ ID NO. 35)[K/R]-[F/Y/W]-[L/I/V]-[L/I/V]-[K/R]; (SEQ ID NO. 36)[K/R]-[Y/W]-[L/I/V]-[L/I/V]-[K/R]; or (SEQ ID NO. 37)[K/R]-F-[L/I/V]-[L/I/V]-[K/R].


4. The method of claim 1, wherein the PCNA interacting motif is selectedfrom the group consisting of SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 and
 26. 5. Themethod of claim 2, wherein the nuclear localization signal sequence isselected from the group consisting of SEQ ID NOs: 75-90 and
 128. 6. Themethod of claim 1, wherein the oligopeptidic compound comprises a PCNAinteracting motif as set forth in SEQ ID NO: 18, a nuclear localizationsignal sequence as set forth in SEQ ID NO: 125 and a cell penetratingsignal sequence as set forth in SEQ ID NO:
 73. 7. The method of claim 1,wherein the oligopeptidic compound comprises a sequence as set forth inSEQ ID NO:
 112. 8. The method of claim 1, wherein said disorder is ahyperproliferative disorder, or said treatment is myeloablation.
 9. Themethod of claim 8, wherein said hyperproliferative disorder is selectedfrom the group consisting of a malignant, pre-malignant or non-malignantneoplastic disorder, an autoimmune disorder, a haematological disorder,a skin disorder, a virally-induced hyperproliferative disorder, amyelodysplastic disorder and a myeloproliferative disorder.
 10. Themethod of claim 8, wherein said hyperproliferative disorder is selectedfrom the group consisting of cancer, benign tumours, psoriaticarthritis, rheumatoid arthritis, inflammatory bowel disease, psoriasis,Reiter's syndrome, pityriasis rubra pilaris, hyperproliferative variantsof the disorders of keratinization, restenosis, diabetic nephropathy,thyroid hyperplasia, Grave's Disease, benign prostatic hypertrophy,Li-Fraumenti syndrome, diabetic retinopathy, peripheral vasculardisease, cervical carcinoma-insitu, familial intestinal polyposes, oralleukoplasias, histiocytoses, keloids, hemangiomas, hyperproliferativearterial stenosis, inflammatory arthritis, hyperkeratoses,papulosquamous eruptions including arthritis, warts, and EBV-induceddisease, scar formation, multiple sclerosis, systemic lupuserythematosus (SLE; lupus), myasthenia gravis, non-malignanthyperplasis, agranuloma, MGUS (Monoclonal Gammopathy of UnknownSignificance), neoplastic meningitis, polycythemia vera, scleromyxedema,papular mucinosis, amyloidosis and Wegener's granulomatosis.
 11. Themethod of claim 1, wherein the disorder is cancer.
 12. The method ofclaim 11, wherein said cancer is selected from the group consisting ofbladder cancer, prostate cancer and a haematological cancer.
 13. Themethod of claim 12, wherein said haematological cancer is multiplemyeloma or leukaemia.
 14. The method of claim 1, wherein saidadministering of said oligopeptidic compound is in conjunction,simultaneously, separately or sequentially with a cytostatic agent. 15.The method of claim 14, wherein said cytostatic agent is an alkylatingagent.
 16. The method of claim 14, wherein said cytostatic agent isselected from the group consisting of actinomycin D, BCNU (carmustine),carboplatin, CCNU, Campothecin (CPT), cantharidin, Cisplatin,cyclophosphamide, cytarabine, dacarbazine, daunorubicin, docetaxel,Doxorubicin, DTIC, epirubicin, Etoposide, gefinitib, gemcitabine,ifosamide, irinotecan, ionomycin, Melphalan, Methotrexate, Mitomycin C(MMC), mitozantronemercaptopurine, Oxaliplatin, Paclitaxel (taxol),PARP-I inhibitor, taxotere, temozolomide (TZM), teniposide, topotecane,treosulfane, vinorelbine, vincristine, vinblastine, 5-Azacytidine,5,6-Dihydro-5-azacytidine and 5-fluorouracil.
 17. A method of treatmentof a disorder or condition comprising: administering an oligopeptidiccompound to a subject; and administering radiotherapy; wherein saidoligopeptidic compound is capable of interacting with proliferating cellnuclear antigen (PCNA), wherein the compound comprises a PCNAinteracting motif which is: (SEQ ID NO. 28)[K/R]-[F/Y/W]-[L/I/V/A/M]-[L/I/V/A/M]-[K/R];

wherein the oligopeptidic compound has 20-50 amino acids and comprises acell penetrating signal sequence selected from the group consisting ofSEQ ID NOs: 38-74, 129, 131 and 133, and wherein in said compound a PCNAinteracting motif is N-terminal to said cell penetrating signalsequence, and wherein said oligopeptidic compound is a sensitizer forsaid radiotherapy.
 18. The method of claim 17, wherein said disorder orcondition is one in which it is desirable to inhibit the growth ofcells.
 19. The method of claim 17, wherein said disorder is ahyperproliferative disorder.
 20. The method of claim 19, wherein saidhyperproliferative disorder is selected from the group consisting of amalignant, pre-malignant or non-malignant neoplastic disorder,inflammation, an autoimmune disorder, a haematological disorder, a skindisorder, a virally-induced hyperproliferative disorder, amyelodysplastic disorder and a myeloproliferative disorder.
 21. Themethod of claim 19, wherein said hyperproliferative disorder is selectedfrom the group consisting of cancer, benign tumours, psoriaticarthritis, rheumatoid arthritis, inflammatory bowel disease, psoriasis,Reiter's syndrome, pityriasis rubra pilaris, hyperproliferative variantsof the disorders of keratinization, restenosis, diabetic nephropathy,thyroid hyperplasia, Grave's Disease, benign prostatic hypertrophy,Li-Fraumenti syndrome, diabetic retinopathy, peripheral vasculardisease, cervical carcinoma-insitu, familial intestinal polyposes, oralleukoplasias, histiocytoses, keloids, hemangiomas, hyperproliferativearterial stenosis, inflammatory arthritis, hyperkeratoses,papulosquamous eruptions including arthritis, warts, and EBV-induceddisease, scar formation, multiple sclerosis, systemic lupuserythematosus (SLE; lupus), myasthenia gravis, non-malignanthyperplasis, agranuloma, MGUS (Monoclonal Gammopathy of UnknownSignificance), neoplastic meningitis, polycythemia vera, scleromyxedema,papular mucinosis, amyloidosis and Wegener's granulomatosis.
 22. Themethod of claim 17, wherein the disorder is cancer.
 23. The method ofclaim 22, wherein said cancer is selected from the group consisting ofbladder cancer, prostate cancer and a haematological cancer.
 24. Themethod of claim 23, wherein said haematological cancer is multiplemyeloma or leukaemia.
 25. The method of claim 17, wherein theoligopeptidic compound further comprises a nuclear localization signalsequence.
 26. The method of claim 17, wherein the PCNA interacting motifis: (SEQ ID NO. 29) [K/R]-[Y/W]-[L/I/V/A/M]-[L/I/V/A/M]-[K/R];(SEQ ID NO. 30) [K/R]-[F/Y/W]-[L/I/V/A]-[L/I/V/A]-[K/R]; (SEQ ID NO. 31)[K/R]-[Y/W]-[L/I/V/A]-[L/I/V/A]-[K/R]; (SEQ ID NO. 32)[K/R]-[F/W]-[L/I/V/A/M]-[L/I/V/A/M]-[K/R]; (SEQ ID NO. 33)[K/R]-[F/W]-[L/I/V/A]-[L/I/V/A]-[K/R]; (SEQ ID NO. 34)[K/R]-[F/W]-[L/I/V]-[L/I/V]-[K/R]; (SEQ ID NO. 35)[K/R]-[F/Y/W]-[L/I/V]-[L/I/V]-[K/R]; (SEQ ID NO. 36)[K/R]-[Y/W]-[L/I/V]-[L/I/V]-[K/R]; or (SEQ ID NO. 37)[K/R]-F-[L/I/V]-[L/I/V]-[K/R].


27. The method of claim 17, wherein the PCNA interacting motif isselected from the group consisting of SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 and 26.28. The method of claim 25, wherein the nuclear localization signalsequence is selected from the group consisting of SEQ ID NOs: 75-90 and128.
 29. The method of claim 17, wherein the oligopeptidic compoundcomprises a PCNA interacting motif as set forth in SEQ ID NO: 18, anuclear localization signal sequence as set forth in SEQ ID NO: 125 anda cell penetrating signal sequence as set forth in SEQ ID NO:
 73. 30.The method of claim 17, wherein the oligopeptidic compound comprises asequence as set forth in SEQ ID NO: 112.